Biosynthesis of human milk oligosaccharides in engineered bacteria

ABSTRACT

The invention provides compositions and methods for engineering bacteria to produce fucosylated oligosaccharides, and the use thereof in the prevention or treatment of infection.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/033,664 filedSep. 23, 2013, now U.S. Pat. No. 9,587,241 issued Mar. 7, 2017, which isa divisional of U.S. Ser. No. 13/398,526 filed Feb. 16, 2012, now U.S.Pat. No. 9,453,230 issued Sep. 27, 2016, and claims the benefit ofpriority under 35 U.S.C. § 119(e) to U.S. Provisional Application No.61/443,470, filed Feb. 16, 2011, the entire contents of each of whichare incorporated herein by reference.

INCORPORATED-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named“37847-505C01US_Sequence_Listing.txt”, which was created on Jan. 26,2017 and is 94 KB in size, are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The invention provides compositions and methods for producing purifiedoligosaccharides, in particular certain fucosylated and/or sialylatedoligosaccharides that are typically found in human milk.

BACKGROUND OF THE INVENTION

Human milk contains a diverse and abundant set of neutral and acidicoligosaccharides (human milk oligosaccharides, HMOS). Many of thesemolecules are not utilized directly by infants for nutrition, but theynevertheless serve critical roles in the establishment of a healthy gutmicrobiome, in the prevention of disease, and in immune function. Priorto the invention described herein, the ability to produce HMOSinexpensively at large scale was problematic. For example, HMOSproduction through chemical synthesis was limited by stereo-specificityissues, precursor availability, product impurities, and high overallcost. As such, there is a pressing need for new strategies toinexpensively manufacture large quantities of HMOS for a variety ofcommercial applications.

SUMMARY OF THE INVENTION

The invention described herein features efficient and economical methodsfor producing fucosylated and sialylated oligosaccharides. The methodfor producing a fucosylated oligosaccharide in a bacterium comprises thefollowing steps: providing a bacterium that comprises a functionalβ-galactosidase gene, an exogenous fucosyltransferase gene, a GDP-fucosesynthesis pathway, and a functional lactose permease gene; culturing thebacterium in the presence of lactose; and retrieving a fucosylatedoligosaccharide from the bacterium or from a culture supernatant of thebacterium.

To produce a fucosylated oligosaccharide by biosynthesis, the bacteriumutilizes an endogenous or exogenous guanosine diphosphate (GDP)-fucosesynthesis pathway. By “GDP-fucose synthesis pathway” is meant a sequenceof reactions, usually controlled and catalyzed by enzymes, which resultsin the synthesis of GDP-fucose. An exemplary GDP-fucose synthesispathway in Escherichia coli is set forth below. In the GDP-fucosesynthesis pathway set forth below, the enzymes for GDP-fucose synthesisinclude: 1) manA=phosphomannose isomerase (PMI), 2)manB=phosphomannomutase (PMM), 3) manC=mannose-1-phosphateguanylyltransferase (GMP), 4) gmd=GDP-mannose-4,6-dehydratase (GMD), 5)fcl=GDP-fucose synthase (GFS), and 6) ΔwcaJ=mutated UDP-glucose lipidcarrier transferase.Glucose→Glc-6-P→Fru-6-P→¹Man-6-P→²Man-1-P→GDP-Man-^(4.5)GDP-Fuc

⁶Colanic acid.

The synthetic pathway from fructose-6-phosphate, a common metabolicintermediate of all organisms, to GDP-fucose consists of 5 enzymaticsteps: 1) PMI (phosphomannose isomerase), 2) PMM (phosphomannomutase),3) GMP (mannose-1-phosphate guanylyltransferase), 4) GMD(GDP-mannose-4,6-dehydratase), and 5) GFS (GDP-fucose synthase).Individual bacterial species possess different inherent capabilitieswith respect to GDP-fucose synthesis. Escherichia coli, for example,contains enzymes competent to perform all five steps, whereas Bacilluslicheniformis is missing enzymes capable of performing steps 4 and 5(i.e., GMD and GFS). Any enzymes in the GDP-synthesis pathway that areinherently missing in any particular bacterial species are provided asgenes on recombinant DNA constructs, supplied either on a plasmidexpression vector or as exogenous genes integrated into the hostchromosome.

The invention described herein details the manipulation of genes andpathways within bacteria such as the enterobacterium Escherichia coliK12 (E. coli) or probiotic bacteria leading to high level synthesis ofHMOS. A variety of bacterial species may be used in the oligosaccharidebiosynthesis methods, for example Erwinia herbicola (Pantoeaagglomerans), Citrobacter freundii, Pantoea citrea, Pectobacteriumcarotovorum, or Xanthomonas campestris. Bacteria of the genus Bacillusmay also be used, including Bacillus subtilis, Bacillus licheniformis,Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus,Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacilluslentus, Bacillus cereus, and Bacillus circulans. Similarly, bacteria ofthe genera Lactobacillus and Lactococcus may be modified using themethods of this invention, including but not limited to Lactobacillusacidophilus, Lactobacillus salivarius, Lactobacillus plantarum,Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillusrhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus,Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri,Lactobacillus jensenii, and Lactococcus lactis. Streptococcusthermophiles and Proprionibacterium freudenreichii are also suitablebacterial species for the invention described herein. Also included aspart of this invention are strains, modified as described here, from thegenera Enterococcus (e.g., Enterococcus faecium and Enterococcusthermophiles), Bifidobacterium (e.g., Bifidobacterium longum,Bifidobacterium infantis, and Bifidobacterium bifidum),Sporolactobacillus spp., Micromomospora spp., Micrococcus spp.,Rhodococcus spp., and Pseudomonas (e.g., Pseudomonas fluorescens andPseudomonas aeruginosa). Bacteria comprising the characteristicsdescribed herein are cultured in the presence of lactose, and afucosylated oligosaccharide is retrieved, either from the bacteriumitself or from a culture supernatant of the bacterium. The fucosylatedoligosaccharide is purified for use in therapeutic or nutritionalproducts, or the bacteria are used directly in such products.

The bacterium also comprises a functional β-galactosidase gene. Theβ-galactosidase gene is an endogenous β-galactosidase gene or anexogenous β-galactosidase gene. For example, the β-galactosidase genecomprises an E. coli lacZ gene (e.g., GenBank Accession Number V00296(GI:41901), incorporated herein by reference). The bacterium accumulatesan increased intracellular lactose pool, and produces a low level ofβ-galactosidase.

A functional lactose permease gene is also present in the bacterium. Thelactose permease gene is an endogenous lactose permease gene or anexogenous lactose permease gene. For example, the lactose permease genecomprises an E. coli lacY gene (e.g., GenBank Accession Number V00295(GI:41897), incorporated herein by reference). Many bacteria possess theinherent ability to transport lactose from the growth medium into thecell, by utilizing a transport protein that is either a homolog of theE. coli lactose permease (e.g., as found in Bacillus licheniformis), ora transporter that is a member of the ubiquitous PTS sugar transportfamily (e.g., as found in Lactobacillus casei and Lactobacillusrhamnosus). For bacteria lacking an inherent ability to transportextracellular lactose into the cell cytoplasm, this ability is conferredby an exogenous lactose transporter gene (e.g., E. coli lacY) providedon recombinant DNA constructs, and supplied either on a plasmidexpression vector or as exogenous genes integrated into the hostchromosome.

The bacterium comprises an exogenous fucosyltransferase gene. Forexample, the exogenous fucosyltransferase gene encodes α(1,2)fucosyltransferase and/or α(1,3) fucosyltransferase. An exemplary α(1,2)fucosyltransferase gene is the wcfW gene from Bacteroides fragilis NCTC9343 (SEQ ID NO: 4). An exemplary α(1,3) fucosyltransferase gene is theHelicobacter pylori 26695 futA gene. One example of the Helicobacterpylori futA gene is presented in GenBank Accession Number HV532291(GI:365791177), incorporated herein by reference.

Alternatively, a method for producing a fucosylated oligosaccharide bybiosynthesis comprises the following steps: providing an entericbacterium that comprises a functional β-galactosidase gene, an exogenousfucosyltransferase gene, a mutation in a colanic acid synthesis gene,and a functional lactose permease gene; culturing the bacterium in thepresence of lactose; and retrieving a fucosylated oligosaccharide fromthe bacterium or from a culture supernatant of the bacterium.

To produce a fucosylated oligosaccharide by biosynthesis, the bacteriumcomprises a mutation in an endogenous colanic acid (a fucose-containingexopolysaccharide) synthesis gene. By “colanic acid synthesis gene” ismeant a gene involved in a sequence of reactions, usually controlled andcatalyzed by enzymes that result in the synthesis of colanic acid.Exemplary colanic acid synthesis genes include an rcsA gene (e.g.,GenBank Accession Number M58003 (GI: 1103316), incorporated herein byreference), an rcsB gene, (e.g., GenBank Accession Number E04821(GI:2173017), incorporated herein by reference), a wcaJ gene, (e.g.,GenBank Accession Number (amino acid) BAA15900 (GI:1736749),incorporated herein by reference), a wzxC gene, (e.g., GenBank AccessionNumber (amino acid) BAA15899 (GI:1736748), incorporated herein byreference), a wcaD gene, (e.g., GenBank Accession Number (amino acid)BAE76573 (GI:85675202), incorporated herein by reference), a wza gene,(e.g., GenBank Accession Number (amino acid) BAE76576 (GI:85675205),incorporated herein by reference), a wzb gene, and (e.g., GenBankAccession Number (amino acid) BAE76575 (GI:85675204), incorporatedherein by reference), and a wzc gene (e.g., GenBank Accession Number(amino acid) BAA15913 (GI:1736763), incorporated herein by reference).

This is achieved through a number of genetic modifications of endogenousE. coli genes involved either directly in colanic acid precursorbiosynthesis, or in overall control of the colanic acid syntheticregulon. Specifically, the ability of the host E. coli strain tosynthesize colanic acid, an extracellular capsular polysaccharide, iseliminated by the deletion of the wcaJ gene, encoding the UDP-glucoselipid carrier transferase. In a wcaJ null background, GDP-fucoseaccumulates in the E. coli cytoplasm. Over-expression of a positiveregulator protein, RcsA, in the colanic acid synthesis pathway resultsin an increase in intracellular GDP-fucose levels. Over-expression of anadditional positive regulator of colanic acid biosynthesis, namely RcsB,is also utilized, either instead of or in addition to over-expression ofRcsA, to increase intracellular GDP-fucose levels. Alternatively,colanic acid biosynthesis is increased following the introduction of anull mutation into the E. coli ion gene (e.g., GenBank Accession NumberL20572 (GI:304907), incorporated herein by reference). Lon is anadenosine-5′-triphosphate (ATP)-dependant intracellular protease that isresponsible for degrading RcsA, mentioned above as a positivetranscriptional regulator of colanic acid biosynthesis in E. coli. In aion null background, RcsA is stabilized, RcsA levels increase, the genesresponsible for GDP-fucose synthesis in E. coli are up-regulated, andintracellular GDP-fucose concentrations are enhanced.

For example, the bacterium further comprises a functional, wild-type E.coli lacZ⁺ gene inserted into an endogenous gene, for example the iongene in E. coli. In this manner, the bacterium may comprise a mutationin a ion gene.

The bacterium also comprises a functional β-galactosidase gene. Theβ-galactosidase gene is an endogenous β-galactosidase gene or anexogenous β-galactosidase gene. For example, the β-galactosidase genecomprises an E. coli lacZ gene. The endogenous lacZ gene of the E. coliis deleted or functionally inactivated, but in such a way thatexpression of the downstream lactose permease (lacY) gene remainsintact.

The bacterium comprises an exogenous fucosyltransferase gene. Forexample, the exogenous fucosyltransferase gene encodes α(1,2)fucosyltransferase and/or α(1,3) fucosyltransferase. An exemplary α(1,2)fucosyltransferase gene is the wcfW gene from Bacteroides fragilis NCTC9343 (SEQ ID NO: 4). An exemplary α(1,3) fucosyltransferase gene is theHelicobacter pylori 26695 futA gene. One example of the Helicobacterpylori futA gene is presented in GenBank Accession Number HV532291(GI:365791177), incorporated herein by reference.

A functional lactose permease gene is also present in the bacterium. Thelactose permease gene is an endogenous lactose permease gene or anexogenous lactose permease gene. For example, the lactose permease genecomprises an E. coli lacY gene.

The bacterium may further comprise an exogenous rcsA and/or rcsB gene(e.g., in an ectopic nucleic acid construct such as a plasmid), and thebacterium optionally further comprises a mutation in a lacA gene (e.g.,GenBank Accession Number X51872 (GI:41891), incorporated herein byreference).

Bacteria comprising the characteristics described herein are cultured inthe presence of lactose, and a fucosylated oligosaccharide is retrieved,either from the bacterium itself or from a culture supernatant of thebacterium. The fucosylated oligosaccharide is purified for use intherapeutic or nutritional products, or the bacteria are used directlyin such products.

The bacteria used herein to produce HMOS are genetically engineered tocomprise an increased intracellular guanosine diphosphate (GDP)-fucosepool, an increased intracellular lactose pool (as compared to wild type)and to comprise fucosyl transferase activity. Accordingly, the bacteriumcontains a mutation in a colanic acid (a fucose-containingexopolysaccharide) synthesis pathway gene, such as a wcaJ gene,resulting in an enhanced intracellular GDP-fucose pool. The bacteriumfurther comprises a functional, wild-type E. coli lacZ⁺ gene insertedinto an endogenous gene, for example the ion gene in E. coli. In thismanner, the bacterium may further comprise a mutation in a ion gene. Theendogenous lacZ gene of the E. coli is deleted or functionallyinactivated, but in such a way that expression of the downstream lactosepermease (lacY) gene remains intact. The organism so manipulatedmaintains the ability to transport lactose from the growth medium, andto develop an intracellular lactose pool for use as an acceptor sugar inoligosaccharide synthesis, while also maintaining a low level ofintracellular beta-galactosidase activity useful for a variety ofadditional purposes. The bacterium may further comprise an exogenousrcsA and/or rcsB gene (e.g., in an ectopic nucleic acid construct suchas a plasmid), and the bacterium optionally further comprises a mutationin a lacA gene. Preferably, the bacterium accumulates an increasedintracellular lactose pool, and produces a low level ofbeta-galactosidase.

The bacterium possesses fucosyl transferase activity. For example, thebacterium comprises one or both of an exogenous fucosyltransferase geneencoding an α(1,2) fucosyltransferase and an exogenousfucosyltransferase gene encoding an α(1,3) fucosyltransferase. Anexemplary α(1,2) fucosyltransferase gene is the wcfW gene fromBacteroides fragilis NCTC 9343 (SEQ ID NO: 4). Prior to the presentinvention, this wcfW gene was not known to encode a protein with anα(1,2) fucosyltransferase activity, and further was not suspected topossess the ability to utilize lactose as an acceptor sugar. Otherα(1,2) fucosyltransferase genes that use lactose as an acceptor sugar(e.g., the Helicobacter pylori 26695 futC gene or the E. coli O128:B12wbsJ gene) may readily be substituted for Bacteroides fragilis wcfW. Oneexample of the Helicobacter pylori futC gene is presented in GenBankAccession Number EF452503 (GI:134142866), incorporated herein byreference.

An exemplary α(1,3) fucosyltransferase gene is the Helicobacter pylori26695 futA gene, although other α(1,3) fucosyltransferase genes known inthe art may be substituted (e.g., α(1,3) fucosyltransferase genes fromHelicobacter hepaticus Hh0072, Helicobacter bilis, Campylobacter jejuni,or from Bacteroides species). The invention includes a nucleic acidconstruct comprising one, two, three or more of the genes describedabove. For example, the invention includes a nucleic acid constructexpressing an exogenous fucosyltransferase gene (encoding α(1,2)fucosyltransferase or α(1,3) fucosyltransferase) transformed into abacterial host strain comprising a deleted endogenous β-galactosidase(e.g., lacZ) gene, a replacement functional β-galactosidase gene of lowactivity, a GDP-fucose synthesis pathway, a functional lactose permeasegene, and a deleted lactose acetyltransferase gene.

Also within the invention is an isolated E. coli bacterium as describedabove and characterized as comprising a defective colanic acid synthesispathway, a reduced level of β-galactosidase (LacZ) activity, and anexogenous fucosyl transferase gene. The invention also includes: a)methods for phenotypic marking of a gene locus in a β-galactosidasenegative host cell by utilizing a β-galactosidase (e.g., lacZ) geneinsert engineered to produce a low but readily detectable level ofβ-galactosidase activity, b) methods for readily detecting lyticbacteriophage contamination in fermentation runs through release anddetection of cytoplasmic β-galactosidase in the cell culture medium, andc) methods for depleting a bacterial culture of residual lactose at theend of production runs. a), b) and c) are each achieved by utilizing afunctional β-galactosidase (e.g., lacZ) gene insert carefully engineeredto direct the expression of a low, but detectable level ofβ-galactosidase activity in an otherwise β-galactosidase negative hostcell.

A purified fucosylated oligosaccharide produced by the methods describedabove is also within the invention. A purified oligosaccharide, e.g.,2′-FL, 3FL, LDFT, is one that is at least 90%, 95%, 98%, 99%, or 100%(w/w) of the desired oligosaccharide by weight.

Purity is assessed by any known method, e.g., thin layer chromatographyor other electrophoretic or chromatographic techniques known in the art.The invention includes a method of purifying a fucosylatedoligosaccharide produced by the genetically engineered bacteriumdescribed above, which method comprises separating the desiredfucosylated oligosaccharide (e.g., 2′-FL) from contaminants in abacterial cell extract or lysate, or bacterial cell culture supernatant.Contaminants include bacterial DNA, protein and cell wall components,and yellow/brown sugar caramels sometimes formed in spontaneous chemicalreactions in the culture medium.

The oligosaccharides are purified and used in a number of products forconsumption by humans as well as animals, such as companion animals(dogs, cats) as well as livestock (bovine, equine, ovine, caprine, orporcine animals, as well as poultry). For example, a pharmaceuticalcomposition comprising purified 2′-fucosyllactose (2′-FL),3-fucosyllactose (3FL), lactodifucotetraose (LDFT), or3′-sialyl-3-fucosyllactose (3′-S3FL) and an excipient is suitable fororal administration. Large quantities of 2′-FL, 3FL, LDFT, or 3′-S3FLare produced in bacterial hosts, e.g., an E. coli bacterium comprising aheterologous α(1,2)fucosyltransferase, a heterologous α(1,3)fucosyltransferase, or a heterologous sialyltransferase, or acombination thereof. An E. coli bacterium comprising an enhancedcytoplasmic pool of each of the following: lactose, GDP-fucose, andCMP-Neu5Ac, is useful in such production systems. In the case of lactoseand GDP-fucose, endogenous E. coli metabolic pathways and genes aremanipulated in ways that result in the generation of increasedcytoplasmic concentrations of lactose and/or GDP-fucose, as compared tolevels found in wild type E. coli. For example, the bacteria contain atleast 10%, 20%, 50%, 2×, 5×, 10× or more of the levels in acorresponding wild type bacteria that lacks the genetic modificationsdescribed above. In the case of CMP-Neu5Ac, endogenous Neu5Ac catabolismgenes are inactivated and exogenous CMP-Neu5Ac biosynthesis genesintroduced into E. coli resulting in the generation of a cytoplasmicpool of CMP-Neu5Ac not found in the wild type bacterium. A method ofproducing a pharmaceutical composition comprising a purified HMOS iscarried out by culturing the bacterium described above, purifying theHMOS produced by the bacterium, and combining the HMOS with an excipientor carrier to yield a dietary supplement for oral administration. Thesecompositions are useful in methods of preventing or treating entericand/or respiratory diseases in infants and adults. Accordingly, thecompositions are administered to a subject suffering from or at risk ofdeveloping such a disease.

The invention therefore provides methods for increasing intracellularlevels of GDP-fucose in Escherichia coli by manipulating the organism'sendogenous colanic acid biosynthesis pathway. This is achieved through anumber of genetic modifications of endogenous E. coli genes involvedeither directly in colanic acid precursor biosynthesis, or in overallcontrol of the colanic acid synthetic regulon. The invention alsoprovides for increasing the intracellular concentration of lactose in E.coli, for cells grown in the presence of lactose, by using manipulationsof endogenous E. coli genes involved in lactose import, export, andcatabolism. In particular, described herein are methods of increasingintracellular lactose levels in E. coli genetically engineered toproduce a human milk oligosaccharide by incorporating a lacA mutationinto the genetically modified E. coli. The lacA mutation prevents theformation of intracellular acetyl-lactose, which not only removes thismolecule as a contaminant from subsequent purifications, but alsoeliminates E. coli's ability to export excess lactose from itscytoplasm, thus greatly facilitating purposeful manipulations of the E.coli intracellular lactose pool.

Also described herein are bacterial host cells with the ability toaccumulate a intracellular lactose pool while simultaneously possessinglow, functional levels of cytoplasmic β-galactosidase activity, forexample as provided by the introduction of a functional recombinant E.coli lacZ gene, or by a β-galactosidase gene from any of a number ofother organisms (e.g., the lac4 gene of Kluyveromyces lactis (e.g.,GenBank Accession Number M84410 (GI: 173304), incorporated herein byreference). Low, functional levels of cytoplasmic β-galactosidaseinclude β-galactosidase activity levels of between 0.05 and 200 units,e.g., between 0.05 and 5 units, between 0.05 and 4 units, between 0.05and 3 units, or between 0.05 and 2 units (for unit definition see:Miller J H, Laboratory CSH. Experiments in molecular genetics. ColdSpring Harbor Laboratory Cold Spring Harbor, N.Y.; 1972; incorporatedherein by reference). This low level of cytoplasmic β-galactosidaseactivity, while not high enough to significantly diminish theintracellular lactose pool, is nevertheless very useful for tasks suchas phenotypic marking of desirable genetic loci during construction ofhost cell backgrounds, for detection of cell lysis due to undesiredbacteriophage contaminations in fermentation processes, or for thefacile removal of undesired residual lactose at the end offermentations.

In one aspect, the human milk oligosaccharide produced by engineeredbacteria comprising an exogenous nucleic acid molecule encoding anα(1,2) fucosyltransferase, is 2′-FL (2′-fucosyllactose). Preferably, theα(1,2)fucosyltransferase utilized is the previously completelyuncharacterized wcfW gene from Bacteroides fragilis NCTC 9343 of thepresent invention, alternatively the futC gene of Helicobacter pylori26695 or the wbsJ gene of E. coli strain O128:B12, or any other α(1,2)fucosyltransferase capable of using lactose as the sugar acceptorsubstrate may be utilized for 2′-FL synthesis. In another aspect thehuman milk oligosaccharide produced by engineered bacteria comprising anexogenous nucleic acid molecule encoding an α(1,3) fucosyltransferase,is 3FL (3-fucosyllactose), wherein the bacterial cell comprises anexogenous nucleic acid molecule encoding an exogenous α(1,3)fucosyltransferase. Preferably, the bacterial cell is E. coli. Theexogenous α(1,3) fucosyltransferase is isolated from, e.g., Helicobacterpylori, H. hepaticus, H. bilis, C. jejuni, or a species of Bacteroides.In one aspect, the exogenous α(1,3) fucosyltransferase comprises H.hepaticus Hh0072, H. pylori 11639 FucTa, or H. pylori UA948 FucTa (e.g.,GenBank Accession Number AF194963 (GI:28436396), incorporated herein byreference). The invention also provides compositions comprising E. coligenetically engineered to produce the human milk tetrasaccharidelactodifucotetraose (LDFT). The E. coli in this instance comprise anexogenous nucleic acid molecule encoding an α(1,2) fucosyltransferaseand an exogenous nucleic acid molecule encoding an α(1,3)fucosyltransferase. In one aspect, the E. coli is transformed with aplasmid expressing an α(1,2) fucosyltransferase and/or a plasmidexpressing an α(1,3) fucosyltransferase. In another aspect, the E. coliis transformed with a plasmid that expresses both an α(1,2)fucosyltransferase and an α(1,3) fucosyltransferase. Alternatively, theE. coli is transformed with a chromosomal integrant expressing an α(1,2)fucosyltransferase and a chromosomal integrant expressing an α(1,3)fucosyltransferase. Optionally, the E. coli is transformed with plasmidpG177.

Also described herein are compositions comprising a bacterial cell thatproduces the human milk oligosaccharide 3′-S3FL(3′-sialyl-3-fucosyllactose), wherein the bacterial cell comprises anexogenous sialyl-transferase gene encoding α(2,3)sialyl-transferase andan exogenous fucosyltransferase gene encoding α(1,3) fucosyltransferase.Preferably, the bacterial cell is E. coli. The exogenousfucosyltransferase gene is isolated from, e.g., Helicobacter pylori, H.hepaticus, H. bilis, C. jejuni, or a species of Bacteroides. Forexample, the exogenous fucosyltransferase gene comprises H. hepaticusHh0072, H. pylori 11639 FucTa, or H. pylori UA948 FucTa. The exogenoussialyltransferase gene utilized for 3′-S3FL production may be obtainedfrom any one of a number of sources, e.g., those described from N.meningitidis and N. gonorrhoeae. Preferably, the bacterium comprises aGDP-fucose synthesis pathway.

Additionally, the bacterium contains a deficient sialic acid catabolicpathway. By “sialic acid catabolic pathway” is meant a sequence ofreactions, usually controlled and catalyzed by enzymes, which results inthe degradation of sialic acid. An exemplary sialic acid catabolicpathway in Escherichia coli is described herein. In the sialic acidcatabolic pathway described herein, sialic acid (Neu5Ac;N-acetylneuraminic acid) is degraded by the enzymes NanA(N-acetylneuraminic acid lyase) and NanK (N-acetylmannosamine kinase).For example, a deficient sialic acid catabolic pathway is engineered inEscherichia coli by way of a null mutation in endogenous nanA(N-acetylneuraminate lyase) (e.g., GenBank Accession Number D00067(GI:216588), incorporated herein by reference) and/or nanK(N-acetylmannosamine kinase) genes (e.g., GenBank Accession Number(amino acid) BAE77265 (GI:85676015), incorporated herein by reference).Other components of sialic acid metabolism include: (nanT) sialic acidtransporter; (ManNAc-6-P) N-acetylmannosamine -6-phosphate; (GlcNAc-6-P)N-acetylglucosamine-6-phosphate; (GlcN-6-P) Glucosamine-6-phosphate; and(Fruc-6-P) Fructose-6-phosphate.

Moreover, the bacterium (e.g., E. coli) also comprises a sialic acidsynthetic capability. For example, the bacterium comprises a sialic acidsynthetic capability through provision of an exogenous UDP-GlcNAc2-epimerase (e.g., neuC of Campylobacter jejuni or equivalent (e.g.,GenBank Accession Number (amino acid) AAG29921 (GI:11095585),incorporated herein by reference)), a Neu5Ac synthase (e.g., neuB of C.jejuni or equivalent, e.g., GenBank Accession Number (amino acid)AAG29920 (GI: 11095584), incorporated herein by reference)), and/or aCMP-Neu5Ac synthetase (e.g., neuA of C. jejuni or equivalent, e.g.,GenBank Accession Number (amino acid) ADN91474 (GI:307748204),incorporated herein by reference).

Additionally, the bacterium also comprises a functional β-galactosidasegene and a functional lactose permease gene. Bacteria comprising thecharacteristics described herein are cultured in the presence oflactose, and a 3′-sialyl-3-fucosyllactose is retrieved, either from thebacterium itself or from a culture supernatant of the bacterium.

Also provided are methods for producing a 3′-sialyl-3-fucosyllactose(3′-S3FL) in an enteric bacterium, wherein the enteric bacteriumcomprises a mutation in an endogenous colanic acid synthesis gene, afunctional lacZ gene, a functional lactose permease gene, an exogenousfucosyltransferase gene encoding α(1,3) fucosyltransferase, and anexogenous sialyltransferase gene encoding an α(2,3)sialyl transferase.Additionally, the bacterium contains a deficient sialic acid catabolicpathway. For example, the bacterium comprises a deficient sialic acidcatabolic pathway by way of a null mutation in endogenous nanA(N-acetylneuraminate lyase) and/or nanK (N-acetylmannosamine kinase)genes. The bacterium also comprises a sialic acid synthetic capability.For example, the bacterium comprises a sialic acid synthetic capabilitythrough provision of an exogenous UDP-GlcNAc 2-epimerase (e.g., neuC ofC. jejuni or equivalent), a Neu5Ac synthase (e.g., neuB of C. jejuni orequivalent), and/or a CMP-Neu5Ac synthetase (e.g., neuA of C. jejuni orequivalent). Bacteria comprising the characteristics described hereinare cultured in the presence of lactose, and a3′-sialyl-3-fucosyllactose is retrieved, either from the bacteriumitself or from a culture supernatant of the bacterium.

Also provided is a method for phenotypic marking of a gene locus in ahost cell, whose native β-galactosidase gene is deleted or inactivated,by utilizing an inserted recombinant β-galactosidase (e.g., lacZ) geneengineered to produce a low, but detectable level of β-galactosidaseactivity. Similarly, the invention also provides methods for depleting abacterial culture of residual lactose in a β-galactosidase negative hostcell, whose native β-galactosidase gene is deleted or inactivated, byutilizing an inserted recombinant β-galactosidase (e.g., lacZ) geneengineered to produce a low but detectable level of β-galactosidaseactivity. Finally, also provided is a method for detecting bacterialcell lysis in a culture of a β-galactosidase negative host cell, whosenative β-galactosidase gene is deleted or inactivated, by utilizing aninserted recombinant β-galactosidase (e.g., lacZ) gene engineered toproduce a low but detectable level of β-galactosidase activity.

Methods of purifying a fucosylated oligosaccharide produced by themethods described herein are carried out by binding the fucosylatedoligosaccharide from a bacterial cell lysate or bacterial cell culturesupernatant of the bacterium to a carbon column, and eluting thefucosylated oligosaccharide from the column. Purified fucosylatedoligosaccharide are produced by the methods described herein.

Optionally, the invention features a vector, e.g., a vector containing anucleic acid. The vector can further include one or more regulatoryelements, e.g., a heterologous promoter. The regulatory elements can beoperably linked to a protein gene, fusion protein gene, or a series ofgenes linked in an operon in order to express the fusion protein. In yetanother aspect, the invention comprises an isolated recombinant cell,e.g., a bacterial cell containing an aforementioned nucleic acidmolecule or vector. The nucleic acid sequence can be optionallyintegrated into the genome.

The term “substantially pure” in reference to a given polypeptide,polynucleotide or oligosaccharide means that the polypeptide,polynucleotide or oligosaccharide is substantially free from otherbiological macromolecules. The substantially pure polypeptide,polynucleotide or oligosaccharide is at least 75% (e.g., at least 80,85, 95, or 99%) pure by dry weight. Purity can be measured by anyappropriate calibrated standard method, for example, by columnchromatography, polyacrylamide gel electrophoresis, thin layerchromatography (TLC) or HPLC analysis.

Polynucleotides, polypeptides, and oligosaccharides of the invention arepurified and/or isolated. Purified defines a degree of sterility that issafe for administration to a human subject, e.g., lacking infectious ortoxic agents. Specifically, as used herein, an “isolated” or “purified”nucleic acid molecule, polynucleotide, polypeptide, protein oroligosaccharide, is substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. For example,Purified HMOS compositions are at least 60% by weight (dry weight) thecompound of interest. Preferably, the preparation is at least 75%, morepreferably at least 90%, and most preferably at least 99%, by weight thecompound of interest. Purity is measured by any appropriate calibratedstandard method, for example, by column chromatography, polyacrylamidegel electrophoresis, thin layer chromatography (TLC) or HPLC analysis.For example, a “purified protein” refers to a protein that has beenseparated from other proteins, lipids, and nucleic acids with which itis naturally associated. Preferably, the protein constitutes at least10, 20, 50 70, 80, 90, 95, 99-100% by dry weight of the purifiedpreparation.

By “isolated nucleic acid” is meant a nucleic acid that is free of thegenes which flank it in the naturally-occurring genome of the organismfrom which the nucleic acid is derived. The term covers, for example:(a) a DNA which is part of a naturally occurring genomic DNA molecule,but is not flanked by both of the nucleic acid sequences that flank thatpart of the molecule in the genome of the organism in which it naturallyoccurs; (b) a nucleic acid incorporated into a vector or into thegenomic DNA of a prokaryote or eukaryote in a manner, such that theresulting molecule is not identical to any naturally occurring vector orgenomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment,a fragment produced by polymerase chain reaction (PCR), or a restrictionfragment; and (d) a recombinant nucleotide sequence that is part of ahybrid gene, i.e., a gene encoding a fusion protein. Isolated nucleicacid molecules according to the present invention further includemolecules produced synthetically, as well as any nucleic acids that havebeen altered chemically and/or that have modified backbones. Forexample, the isolated nucleic acid is a purified cDNA or RNApolynucleotide.

A “heterologous promoter”, when operably linked to a nucleic acidsequence, refers to a promoter which is not naturally associated withthe nucleic acid sequence.

The terms “express” and “over-express” are used to denote the fact that,in some cases, a cell useful in the method herein may inherently expresssome of the factor that it is to be genetically altered to produce, inwhich case the addition of the polynucleotide sequence results inover-expression of the factor. That is, more factor is expressed by thealtered cell than would be, under the same conditions, by a wild typecell. Similarly, if the cell does not inherently express the factor thatit is genetically altered to produce, the term used would be to merely“express” the factor since the wild type cell did not express the factorat all.

The terms “treating” and “treatment” as used herein refer to theadministration of an agent or formulation to a clinically symptomaticindividual afflicted with an adverse condition, disorder, or disease, soas to effect a reduction in severity and/or frequency of symptoms,eliminate the symptoms and/or their underlying cause, and/or facilitateimprovement or remediation of damage. The terms “preventing” and“prevention” refer to the administration of an agent or composition to aclinically asymptomatic individual who is susceptible to a particularadverse condition, disorder, or disease, and thus relates to theprevention of the occurrence of symptoms and/or their underlying cause.

The invention provides a method of treating, preventing, or reducing therisk of infection in a subject comprising administering to said subjecta composition comprising a human milk oligosaccharide, purified from aculture of a recombinant strain of the current invention, wherein theHMOS binds to a pathogen and wherein the subject is infected with or atrisk of infection with the pathogen. In one aspect, the infection iscaused by a Norwalk-like virus or Campylobacter jejuni. The subject ispreferably a mammal in need of such treatment. The mammal is, e.g., anymammal, e.g., a human, a primate, a mouse, a rat, a dog, a cat, a cow, ahorse, or a pig. In a preferred embodiment, the mammal is a human. Forexample, the compositions are formulated into animal feed (e.g.,pellets, kibble, mash) or animal food supplements for companion animals,e.g., dogs or cats, as well as livestock or animals grown for foodconsumption, e.g., cattle, sheep, pigs, chickens, and goats. Preferably,the purified HMOS is formulated into a powder (e.g., infant formulapowder or adult nutritional supplement powder, each of which is mixedwith a liquid such as water or juice prior to consumption) or in theform of tablets, capsules or pastes or is incorporated as a component indairy products such as milk, cream, cheese, yogurt or kefir, or as acomponent in any beverage, or combined in a preparation containing livemicrobial cultures intended to serve as probiotics, or in prebioticpreparations intended to enhance the growth of beneficial microorganismseither in vitro or in vivo. For example, the purified sugar (e.g.,2′-FL) can be mixed with a Bifidobacterium or Lactobacillus in aprobiotic nutritional composition. (i.e. Bifidobacteria are beneficialcomponents of a normal human gut flora and are also known to utilizeHMOS for growth.

By the terms “effective amount” and “therapeutically effective amount”of a formulation or formulation component is meant a nontoxic butsufficient amount of the formulation or component to provide the desiredeffect.

The transitional term “comprising,” which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, unrecited elements or methodsteps. By contrast, the transitional phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. Thetransitional phrase “consisting essentially of” limits the scope of aclaim to the specified materials or steps “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described below. All publishedforeign patents and patent applications cited herein are incorporatedherein by reference. Genbank and NCBI submissions indicated by accessionnumber cited herein are incorporated herein by reference. All otherpublished references, documents, manuscripts and scientific literaturecited herein are incorporated herein by reference. In the case ofconflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration showing the synthetic pathway of themajor neutral fucosyl-oligosaccharides found in human milk.

FIG. 2 is a schematic illustration showing the synthetic pathway of themajor sialyloligosaccharides found in human milk.

FIG. 3 is a schematic demonstrating metabolic pathways and the changesintroduced into them to engineer 2′-fucosyllactose (2′-FL) synthesis inEscherichia coli (E. coli). Specifically, the lactose synthesis pathwayand the GDP-fucose synthesis pathway are illustrated. In the GDP-fucosesynthesis pathway: manA=phosphomannose isomerase (PMI),manB=phosphomannomutase (PMM), manC=mannose-1-phosphateguanylyltransferase (GMP), gmd=GDP-mannose-4,6-dehydratase,fcl=GDP-fucose synthase (GFS), and ΔwcaJ=mutated UDP-glucose lipidcarrier transferase.

FIG. 4 is a photograph of a thin layer chromatogram of purified 2′-FLproduced in E. coli.

FIG. 5 is a schematic demonstrating metabolic pathways and the changesintroduced into them to engineer 3′-sialyllactose (3′-SL) synthesis inE. coli. Abbreviations include: (Neu5Ac) N-acetylneuraminic acid, sialicacid; (nanT) sialic acid transporter; (ΔnanA) mutated N-acetylneuraminicacid lyase; (ManNAc) N-acetylmannosamine; (ΔnanK) mutatedN-acetylmannosamine kinase; (ManNAc-6-P)N-acetylmannosamine-6-phosphate; (GlcNAc -6-P)N-acetylglucosamine-6-phosphate; (GlcN-6-P) Glucosamine-6-phosphate;(Fruc-6-P) Fructose-6-phosphate; (neuA), CMP-N-acetylneuraminic acidsynthetase; (CMP-Neu5Ac) CMP-N-acetylneuraminic acid; and (neuB),N-acetylneuraminic acid synthase.

FIG. 6 is a schematic demonstrating metabolic pathways and the changesintroduced into them to engineer 3-fucosyllactose (3-FL) synthesis in E.coli.

FIG. 7 is a plasmid map of pG175, which expresses the E. coliα(1,2)fucosyltransferase gene wbsJ.

FIG. 8 is a photograph of a western blot of lysates of E. colicontaining pG175 and expressing wbsJ, and of cells containing pG171, apG175 derivative plasmid carrying the H. pylori 26695 futC gene in placeof wbsJ and which expresses futC.

FIG. 9 is a photograph of a thin layer chromatogram of 3FL produced inE. coli containing the plasmid pG176 and induced for expression of theH. pylori 26695 α(1,3)fucosyltransferase gene futA by tryptophanaddition.

FIG. 10 is a plasmid map of pG177, which contains both the H. pylori26695 α(1,2)fucosyltransferase gene futC and the H. pylori 26695α(1,3)fucosyltransferase gene futA, configured as an operon.

FIG. 11 is a photograph of a thin layer chromatogram of 2′-FL, 3FL, andLDFT (lactodifucotetraose) produced in E. coli, directed by plasmidspG171, pG175 (2′-FL), pG176 (3FL), and pG177 (LDFT, 2′-FL and 3FL).

FIG. 12 is a diagram showing the replacement of the ion gene in E. colistrain E390 by a DNA fragment carrying both a kanamycin resistance gene(derived from transposon Tn5) and a wild-type E. coli lacZ+ codingsequence.

FIG. 13A-E is a DNA sequence with annotations (in GenBank format) of theDNA insertion into the ion region diagrammed in FIG. 12 (SEQ ID NOs9-15).

FIG. 14 is a table containing the genotypes of several E. coli strainsof the current invention.

FIG. 15 is a plasmid map of pG186, which expresses theα(1,2)fucosyltransferase gene futC in an operon with the colanic acidpathway transcription activator gene rcsB.

FIG. 16 is a photograph of a western blot of lysates of E. colicontaining pG180, a pG175 derivative plasmid carrying the B. fragiliswcfW gene in place of wbsJ and which expresses wcfW, and of cellscontaining pG171, a pG175 derivative plasmid carrying the H. pylori26695 futC gene in place of wbsJ and which expresses futC.

FIG. 17 is a photograph of a thin layer chromatogram of 2′-FL producedin E. coli by cells carrying plasmids pG180 or pG171 and induced forexpression of wcfW or futC respectively.

FIG. 18 is a photograph of a thin layer chromatogram showing thekinetics and extent of 2′-FL production in a 10 L bioreactor of E. colihost strain E403 transformed with plasmid pG171.

FIG. 19 is a column chromatogram and a TLC analysis of the resolution ona carbon column of a sample of 2′-FL made in E. coli from a lactoseimpurity.

FIG. 20 is a photograph of a thin layer chromatogram showing 3′-SL inculture medium produced by E. coli strain E547, containing plasmidsexpressing a bacterial α(2,3)sialyltransferase and neuA, neuB and neuC.

DETAILED DESCRIPTION OF THE INVENTION

Human milk glycans, which comprise both oligosaccharides (HMOS) andtheir glycoconjugates, play significant roles in the protection anddevelopment of human infants, and in particular the infantgastrointestinal (GI) tract. Milk oligosaccharides found in variousmammals differ greatly, and their composition in humans is unique(Hamosh M., 2001 Pediatr Clin North Am, 48:69-86; Newburg D. S., 2001Adv Exp Med Biol, 501:3-10). Moreover, glycan levels in human milkchange throughout lactation and also vary widely among individuals(Morrow A. L. et al., 2004 J Pediatr, 145:297-303; Chaturvedi P et al.,2001 Glycobiology, 11:365-372). Previously, a full exploration of theroles of HMOS was limited by the inability to adequately characterizeand measure these compounds. In recent years sensitive and reproduciblequantitative methods for the analysis of both neutral and acidic HMOShave been developed (Erney, R., Hilty, M., Pickering, L., Ruiz-Palacios,G., and Prieto, P. (2001) Adv Exp Med Biol 501, 285-297. Bao, Y., andNewburg, D. S. (2008) Electrophoresis 29, 2508-2515). Approximately 200distinct oligosaccharides have been identified in human milk, andcombinations of a small number of simple epitopes are responsible forthis diversity (Newburg D. S., 1999 Curr Med Chem, 6:117-127; NinonuevoM. et al., 2006 J Agric Food Chem, 54:7471-74801). HMOS are composed of5 monosaccharides: D-glucose (Glc), D-galactose (Gal),N-acetylglucosamine (GlcNAc), L-fucose (Fuc), and sialic acid (N-acetylneuraminic acid, Neu5Ac, NANA). HMOS are usually divided into two groupsaccording to their chemical structures: neutral compounds containingGlc, Gal, GlcNAc, and Fuc, linked to a lactose (Galβ1-4Glc) core, andacidic compounds including the same sugars, and often the same corestructures, plus NANA (Charlwood J. et al., 1999 Anal Biochem,273:261-277; Martifn-Sosa et al., 2003 J Dairy Sci, 86:52-59; ParkkinenJ. and Finne J., 1987 Methods Enzymol, 138:289-300; Shen Z. et al., 2001J Chromatogr A, 921:315-321). Approximately 70-80% of oligosaccharidesin human milk are fucosylated, and their synthetic pathways are believedto proceed in a manner similar to those pathways shown in FIG. 1 (withthe Type I and Type II subgroups beginning with different precursormolecules). A smaller proportion of the oligosaccharides in human milkare sialylated, or are both fucosylated and sialylated. FIG. 2 outlinespossible biosynthetic routes for sialylated (acidic) HMOS, althoughtheir actual synthetic pathways in humans are not yet completelydefined.

Interestingly, HMOS as a class, survive transit through the intestine ofinfants very efficiently, a function of their being poorly transportedacross the gut wall and of their resistance to digestion by human gutenzymes (Chaturvedi, P., Warren, C. D., Buescher, C. R., Pickering, L.K. & Newburg, D. S. Adv Exp Med Biol 501, 315-323 (2001)). Oneconsequence of this survival in the gut is that HMOS are able tofunction as prebiotics, i.e. they are available to serve as an abundantcarbon source for the growth of resident gut commensal microorganisms(Ward, R. E., Nifionuevo, M., Mills, D. A., Lebrilla, C. B., and German,J. B. (2007) Mol Nutr Food Res 51, 1398-1405). Recently, there isburgeoning interest in the role of diet and dietary prebiotic agents indetermining the composition of the gut microflora, and in understandingthe linkage between the gut microflora and human health (Roberfroid, M.,Gibson, G. R., Hoyles, L, McCartney, A. L., Rastall, R., Rowland, I.,Wolvers, D., Watzl, B., Szajewska, H., Stahl, B., Guarner, F.,Respondek, F., Whelan, K., Coxam, V., Davicco, M. J., Léotoing, L.,Wittrant, Y., Delzenne, N. M., Cani, P. D., Neyrinck, A. M., andMeheust, A. (2010) Br J Nutr 104 Suppl 2, S1-63).

A number of human milk glycans possess structural homology to cellreceptors for enteropathogens, and serve roles in pathogen defense byacting as molecular receptor “decoys”. For example, pathogenic strainsof Campylobacter bind specifically to glycans in human milk containingthe H-2 epitope, i.e., 2′-fucosyl-N-acetyllactosamine or2′-fucosyllactose (2′-FL); Campylobacter binding and infectivity areinhibited by 2′-FL and other glycans containing this H-2 epitope(Ruiz-Palacios, G. M., Cervantes, L. E., Ramos, P., Chavez-Munguia, B.,and Newburg, D. S. (2003) J Biol Chem 278, 14112-14120). Similarly, somediarrheagenic E. coli pathogens are strongly inhibited in vivo by HMOScontaining 2′-linked fucose moieties. Several major strains of humancaliciviruses, especially the noroviruses, also bind to 2′-linkedfucosylated glycans, and this binding is inhibited by human milk2′-linked fucosylated glycans. Consumption of human milk that has highlevels of these 2′-linked fucosyloligosaccharides has been associatedwith lower risk of norovirus, Campylobacter, ST of E. coli-associateddiarrhea, and moderate-to-severe diarrhea of all causes in a Mexicancohort of breastfeeding children (Newburg D. S. et al., 2004Glycobiology, 14:253-263; Newburg D. S. et al., 1998 Lancet,351:1160-1164). Several pathogens are also known to utilize sialylatedglycans as their host receptors, such as influenza (Couceiro, J. N.,Paulson, J. C. & Baum, L G. Virus Res 29, 155-165 (1993)), parainfluenza(Amonsen, M., Smith, D. F., Cummings, R. D. & Air, G. M. J Virol 81,8341-8345 (2007), and rotoviruses (Kuhlenschmidt, T. B., Hanafin, W. P.,Gelberg, H. B. & Kuhlenschmidt, M. S. Adv Exp Med Biol 473, 309-317(1999)). The sialyl-Lewis X epitope is used by Helicobacter pylori(Mahdavi, J., Sondén, B., Hurtig, M., Olfat, F. O., et al. Science 297,573-578 (2002)), Pseudomonas aeruginosa (Scharfman, A., Delmotte, P.,Beau, J., Lamblin, G., et al. Glycoconj J 17, 735-740 (2000)), and somestrains of noroviruses (Rydell, G. E., Nilsson, J., Rodriguez-Diaz, J.,Ruvoën-Clouet, N., et al. Glycobiology 19, 309-320 (2009)).

While studies suggest that human milk glycans could be used asprebiotics and as antimicrobial anti-adhesion agents, the difficulty andexpense of producing adequate quantities of these agents of a qualitysuitable for human consumption has limited their full-scale testing andperceived utility. What has been needed is a suitable method forproducing the appropriate glycans in sufficient quantities at reasonablecost. Prior to the invention described herein, there were attempts touse several distinct synthetic approaches for glycan synthesis. Novelchemical approaches can synthesize oligosaccharides (Flowers, H. M.Methods Enzymol 50, 93-121 (1978); Seeberger, P. H. Chem Commun (Camb)1115-1121 (2003)), but reactants for these methods are expensive andpotentially toxic (Koeller, K. M. & Wong, C. H. Chem Rev 100, 4465-4494(2000)). Enzymes expressed from engineered organisms (Albermann, C.,Piepersberg, W. & Wehmeier, U. F. Carbohydr Res 334, 97-103 (2001);Bettler, E., Samain, E., Chazalet, V., Bosso, C., et al. Glycoconj J 16,205-212 (1999); Johnson, K. F. Glycoconj J 16, 141-146 (1999); Palcic,M. M. Curr Opin Biotechnol 10, 616-624 (1999); Wymer, N. & Toone, E. J.Curr Opin Chem Biol 4, 110-119 (2000)) provide a precise and efficientsynthesis (Palcic, M. M. Curr Opin Biotechnol 10, 616-624 (1999));Crout, D. H. & Vic, G. Curr Opin Chem Biol 2, 98-111 (1998)), but thehigh cost of the reactants, especially the sugar nucleotides, limitstheir utility for low-cost, large-scale production. Microbes have beengenetically engineered to express the glycosyltransferases needed tosynthesize oligosaccharides from the bacteria's innate pool ofnucleotide sugars (Endo, T., Koizumi, S., Tabata, K., Kakita, S. &Ozaki, A. Carbohydr Res 330, 439-443 (2001); Endo, T., Koizumi, S.,Tabata, K. & Ozaki, A. Appl Microbiol Biotechnol 53, 257-261 (2000);Endo, T. & Koizumi, S. Curr Opin Struct Biol 10, 536-541 (2000); Endo,T., Koizumi, S., Tabata, K., Kakita, S. & Ozaki, A. Carbohydr Res 316,179-183 (1999); Koizumi, S., Endo, T., Tabata, K. & Ozaki, A. NatBiotechnol 16, 847-850 (1998)). However, low overall product yields andhigh process complexity have limited the commercial utility of theseapproaches.

Prior to the invention described herein, which enables the inexpensiveproduction of large quantities of neutral and acidic HMOS, it had notbeen possible to fully investigate the ability of this class of moleculeto inhibit pathogen binding, or indeed to explore their full range ofpotential additional functions.

Prior to the invention described herein, chemical syntheses of HMOS werepossible, but were limited by stereo-specificity issues, precursoravailability, product impurities, and high overall cost (Flowers, H. M.Methods Enzymol 50, 93-121 (1978); Seeberger, P. H. Chem Commun (Camb)1115-1121 (2003); Koeller, K. M. & Wong, C. H. Chem Rev 100, 4465-4494(2000)). Also, prior to the invention described herein, in vitroenzymatic syntheses were also possible, but were limited by arequirement for expensive nucleotide-sugar precursors. The inventionovercomes the shortcomings of these previous attempts by providing newstrategies to inexpensively manufacture large quantities of human milkoligosaccharides for use as dietary supplements. The invention describedherein makes use of an engineered bacterium E. coli (or other bacteria)engineered to produce 2′-FL, 3FL, LDFT, or sialylatedfucosyl-oligosaccharides in commercially viable levels, for example themethods described herein enable the production of 2′-fucosylactoseat >50 g/L in bioreactors.

Example 1. Engineering of E. coli to Generate Host Strains for theProduction of Fucosylated Human Milk Oligosaccharides

The E. coli K12 prototroph W3110 was chosen as the parent background forfucosylated HMOS biosynthesis. This strain had previously been modifiedat the ampC locus by the introduction of a tryptophan-inducibleP_(trpB)-cI+ repressor construct (McCoy, J. & Lavallie, E. Currentprotocols in molecular biology/edited by Frederick M. Ausubel . . . [etal.](2001)), enabling economical production of recombinant proteins fromthe phage λP_(L) promoter (Sanger, F., Coulson, A. R., Hong, G. F.,Hill, D. F. & Petersen, G. B. J Mol Biol 162, 729-773 (1982)) throughinduction with millimolar concentrations of tryptophan (Mieschendahl,M., Petri, T. & Hänggi, U. Nature Biotechnology 4, 802-808 (1986)). Thestrain GI724, an E. coli W3110 derivative containing thetryptophan-inducible P_(trpB)-cI+ repressor construct in ampC, was usedat the basis for further E. coli strain manipulations (FIG. 14).

Biosynthesis of fucosylated HMOS requires the generation of an enhancedcellular pool of both lactose and GDP-fucose (FIG. 3). This enhancementwas achieved in strain GI724 through several manipulations of thechromosome using) Red recombineering (Court, D. L, Sawitzke, J. A. &Thomason, L. C. Annu Rev Genet 36, 361-388 (2002)) and generalized P1phage transduction (Thomason, L. C., Costantino, N. & Court, D. L. MolBiol Chapter 1, Unit 1.17 (2007)). FIG. 14 is a table presenting thegenotypes of several E. coli strains constructed for this invention. Theability of the E. coli host strain to accumulate intracellular lactosewas first engineered in strain E183 (FIG. 14) by simultaneous deletionof the endogenous β-galactosidase gene (lacZ) and the lactose operonrepressor gene (lacI). During construction of this deletion in GI724 toproduce E183, the lacIq promoter was placed immediately upstream of thelactose permease gene, lacY. The modified strain thus maintains itsability to transport lactose from the culture medium (via LacY), but isdeleted for the wild-type copy of the lacZ (β-galactosidase) generesponsible for lactose catabolism. An intracellular lactose pool istherefore created when the modified strain is cultured in the presenceof exogenous lactose.

Subsequently, the ability of the host E. coli strain to synthesizecolanic acid, an extracellular capsular polysaccharide, was eliminatedin strain E205 (FIG. 14) by the deletion of the wcaJ gene, encoding theUDP-glucose lipid carrier transferase (Stevenson, G., Andrianopoulos,K., Hobbs, M. & Reeves, P. R. J Bacteriol 178, 4885-4893 (1996)) instrain E183. In a wcaJ null background, GDP-fucose accumulates in the E.coli cytoplasm (Dumon, C., Priem, B., Martin, S. L., Heyraud, A., et al.Glycoconj J 18, 465-474 (2001)).

A thyA (thymidylate synthase) mutation was introduced into strain E205to produce strain E214 (FIG. 14) by P1 transduction. In the absence ofexogenous thymidine, thyA strains are unable to make DNA, and die. Thedefect can be complemented in trans by supplying a wild-type thyA geneon a multicopy plasmid (Belfort, M., Maley, G. F. & Maley, F.Proceedings of the National Academy of Sciences 80, 1858 (1983)). Thiscomplementation is used herein as a means of plasmid maintenance(eliminating the need for a more conventional antibiotic selectionscheme to maintain plasmid copy number).

One strategy for GDP-fucose production is to enhance the bacterialcell's natural synthesis capacity. For example, this is enhancement isaccomplished by inactivating enzymes involved in GDP-fucose consumption,and/or by overexpressing a positive regulator protein, RcsA, in thecolanic acid (a fucose-containing exopolysaccharide) synthesis pathway.Collectively, this metabolic engineering strategy re-directs the flux ofGDP-fucose destined for colanic acid synthesis to oligosaccharidesynthesis (FIG. 3). By “GDP-fucose synthesis pathway” is meant asequence of reactions, usually controlled and catalyzed by enzymes,which results in the synthesis of GDP-fucose. An exemplary GDP-fucosesynthesis pathway in Escherichia coli as described in FIG. 3 is setforth below. In the GDP-fucose synthesis pathway set forth below, theenzymes for GDP-fucose synthesis include: 1) manA=phosphomannoseisomerase (PMI), 2) manB=phosphomannomutase (PMM), 3)manC=mannose-1-phosphate guanylyltransferase (GMP), 4)gmd=GDP-mannose-4,6-dehydratase (GMD), 5) fcl=GDP-fucose synthase (GFS),and 6) ΔwcaJ=mutated UDP-glucose lipid carrier transferase.Glucose→Glc-6-P→Fru-6-P→¹Man-6-P→²Man-1-P→GDP-Man-^(4.5)GDP-Fuc

⁶Colanic acid.

Specifically, the magnitude of the cytoplasmic GDP-fucose pool in strainE214 is enhanced by over-expressing the E. coli positive transcriptionalregulator of colanic acid biosynthesis, RscA (Gottesman, S. & Stout, V.Mol Microbiol 5, 1599-1606 (1991)). This over-expression of RcsA isachieved by incorporating a wild-type rcsA gene, including its promoterregion, onto a multicopy plasmid vector and transforming the vector intothe E. coli host, e.g. into E214. This vector typically also carriesadditional genes, in particular one or two fucosyltransferase genesunder the control of the pL promoter, and thyA and beta-lactamase genesfor plasmid selection and maintenance. pG175 (SEQ ID NO: 1 and FIG. 7),pG176 (SEQ ID NO: 2), pG177 (SEQ ID NO: 3 and FIG. 10), pG171 (SEQ IDNO: 5) and pG180 (SEQ ID NO: 6) are all examples offucosyltransferase-expressing vectors that each also carry a copy of thercsA gene, for the purpose of increasing the intracellular GDP-fucosepool of the E. coli hosts transformed with these plasmids.Over-expression of an additional positive regulator of colanic acidbiosynthesis, namely RcsB (Gupte G, Woodward C, Stout V. Isolation andcharacterization of rcsb mutations that affect colanic acid capsulesynthesis in Escherichia coli K-12. J Bacteriol 1997, July;179(13):4328-35.), can also be utilized, either instead of or inaddition to over-expression of RcsA, to increase intracellularGDP-fucose levels. Over-expression of rcsB is also achieved by includingthe gene on a multi-copy expression vector. pG186 is such a vector (SEQID NO: 8 and FIG. 15). pG186 expresses rcsB in an operon with futC underpL promoter control. The plasmid also expresses rcsA, driven off its ownpromoter. pG186 is a derivative of pG175 in which the α(1,2) FT (wbsJ)sequence is replaced by the H. pylori futC gene (FutC is MYC-tagged atits C-terminus). In addition, at the XhoI restriction site immediately3′ of the futC CDS, the E. coli rcsB gene is inserted, complete with aribosome binding site at the 5′ end of the rcsB CDS, and such that futCand rcsB form an operon.

A third means to increase the intracellular GDP-fucose pool may also beemployed. Colanic acid biosynthesis is increased following theintroduction of a null mutation into the E. coli ion gene. Lon is anATP-dependant intracellular protease that is responsible for degradingRcsA, mentioned above as a positive transcriptional regulator of colanicacid biosynthesis in E. coli (Gottesman, S. & Stout, V. Mol Microbiol 5,1599-1606 (1991)). In a ion null background, RcsA is stabilized, RcsAlevels increase, the genes responsible for GDP-fucose synthesis in E.coli are up-regulated, and intracellular GDP-fucose concentrations areenhanced. The ion gene was almost entirely deleted and replaced by aninserted functional, wild-type, but promoter-less E. coli lacZ⁺ gene(Δlon::(kan, lacZ⁺) in strain E214 to produce strain E390. λRedrecombineering was used to perform the construction. FIG. 12 illustratesthe new configuration of genes engineered at the ion locus in E390. FIG.13A-E presents the complete DNA sequence of the region, with annotationsin GenBank format. Genomic DNA sequence surrounding the lacZ+ insertioninto the Ion region in E. coli strain E390 is set forth below (SEQ IDNO: 7) The ion mutation in E390 increases intracellular levels of RcsA,and enhances the intracellular GDP-fucose pool. The inserted lacZ⁺cassette not only knocks out ion, but also converts the lacZ⁻ host backto both a lacZ⁺ genotype and phenotype. The modified strain produces aminimal (albeit still readily detectable) level of β-galactosidaseactivity (1-2 units), which has very little impact on lactoseconsumption during production runs, but which is useful in removingresidual lactose at the end of runs, is an easily scorable phenotypicmarker for moving the ion mutation into other lacZ E. coli strains by P1transduction, and can be used as a convenient test for cell lysis (e.g.caused by unwanted bacteriophage contamination) during production runsin the bioreactor.

The production host strain, E390 incorporates all the above geneticmodifications and has the following genotype:ampC::(P_(trpB)λcI⁺),P_(lacI) _(q)(ΔlacI-lacZ)₁₅₈lacY⁺,ΔwcaJ,thyA₇₄₈::Tn10,Δlon::(kan,lacZ⁺)

An additional modification of E390 that is useful for increasing thecytoplasmic pool of free lactose (and hence the final yield of 2′-FL) isthe incorporation of a lacA mutation. LacA is a lactoseacetyltransferase that is only active when high levels of lactoseaccumulate in the E. coli cytoplasm. High intracellular osmolarity(e.g., caused by a high intracellular lactose pool) can inhibitbacterial growth, and E. coli has evolved a mechanism for protectingitself from high intra cellular osmolarity caused by lactose by“tagging” excess intracellular lactose with an acetyl group using LacA,and then actively expelling the acetyl-lactose from the cell (Danchin,A. Bioessays 31, 769-773 (2009)). Production of acetyl-lactose in E.coli engineered to produce 2′-FL or other human milk oligosaccharides istherefore undesirable: it reduces overall yield. Moreover,acetyl-lactose is a side product that complicates oligosaccharidepurification schemes. The incorporation of a lacA mutation resolvesthese problems. Strain E403 (FIG. 14) is a derivative of E390 thatcarries a deletion of the lacA gene and thus is incapable ofsynthesizing acetyl-lactose.

The production host strain, E403 incorporates all the above geneticmodifications and has the following genotype:ampC::(P_(trpB)λcI⁺),P_(lacI) _(q)(ΔlacI-lacZ)₁₅₈lacY⁺,ΔwcaJ,thyA₇₄₈::Tn10,Δlon::(kan,lacZ⁺)ΔlacA

Example 2. 2′-FL Production at Small Scale

Various alternative α(1,2) fucosyltransferases are able to utilizelactose as a sugar acceptor and are available for the purpose of 2′-FLsynthesis when expressed under appropriate culture conditions in E. coliE214, E390 or E403. For example the plasmid pG175 (ColE1, thyA+, bla+,P_(L2)-wbsJ, rcsA+) (SEQ ID NO: 1, FIG. 7) carries the wbsJα(1,2)fucosyltransferase gene of E. coli strain O128:B12 and can directthe production of 2′-FL in E. coli strain E403. In another exampleplasmid pG171 (ColE1, thyA+, bla+, P_(L2)-futC, rcsA+) (SEQ ID NO: 5),carries the H. pylori 26695 futC α(1,2)fucosyltransferase gene (Wang,G., Rasko, D. A., Sherburne, R. & Taylor, D. E. Mol Microbiol 31,1265-1274 (1999)) and will also direct the production of 2′-FL in strainE403. In a preferred example, the plasmid pG180 (ColE1, thyA+, bla+,P_(L2)-wcfW, rcsA+) (SEQ ID NO: 6) carries the previouslyuncharacterized Bacteriodes fragilis NCTC 9343 wcfWα(1,2)fucosyltransferase gene of the current invention and directs theproduction of 2′-FL in E. coli strain E403.

The addition of tryptophan to the lactose-containing growth medium ofcultures of any one of the strains E214, E390 or E403, when transformedwith any one of the plasmids pG171, pG175 or pG180 leads, for eachparticular strain/plasmid combination, to activation of the host E. colitryptophan utilization repressor TrpR, subsequent repression of P_(trpB)and a consequent decrease in cytoplasmic cI levels, which results in ade-repression of P_(L), expression of futC, wbsJ or wcfW, respectively,and production of 2′-FL. FIG. 8 is a coomassie blue-stained SDS PAGE gelof lysates of E. coli containing pG175 and expressing wbsJ, and of cellscontaining pG171 and expressing futC. Prominent stained protein bandsrunning at a molecular weight of approximately 35 kDa are seen for bothWbsJ and FutC at 4 and 6 h following P_(L) induction (i.e., afteraddition of tryptophan). FIG. 16 is a coomassie blue-stained SDS PAGEgel of lysates of E. coli containing pG180 and expressing wcfW, and ofcells containing pG171 and expressing H. pylori futC. Prominent stainedbands for both WcfW and FutC are seen at a molecular weight ofapproximately 40 kDa at 4 and 6 h following P_(L) induction (i.e., afteraddition of tryptophan to the growth medium). For 2′-FL production insmall scale laboratory cultures (<100 ml) strains were grown at 30 C ina selective medium lacking both thymidine and tryptophan to earlyexponential phase (e.g. M9 salts, 0.5% glucose, 0.4% casaminoacids).Lactose was then added to a final concentration of 0.5 or 1%, along withtryptophan (200 μM final) to induce expression of the α(1,2)fucosyltransferase, driven from the P_(L) promoter. At the end of theinduction period (˜24 h) TLC analysis was performed on aliquots ofcell-free culture medium, or of heat extracts of cells (treatments at 98C for 10 min, to release sugars contained within the cell). FIG. 11shows a TLC analysis of cytoplasmic extracts of engineered E. coli cellstransformed with pG175 or pG171. Cells were induced to express wbsJ orfutC, respectively, and grown in the presence of lactose. The productionof 2′-FL can clearly be seen in heat extracts of cells carrying eitherplasmid. FIG. 17 shows a TLC analysis of cytoplasmic extracts ofengineered E. coli cells transformed with pG180 or pG171. Cells wereinduced to express wcfW or futC, respectively, and grown in the presenceof lactose. The production of 2′-FL can clearly be seen with bothplasmids. Prior to the present invention the wcfW gene had never beenshown to encode a protein with demonstrated α(1,2) fucosyltransferaseactivity, or to utilize lactose as a sugar acceptor substrate.

The DNA sequence of the Bacteroides fragilis strain NCTC 9343 wcfW gene(protein coding sequence) is set forth below (SEQ ID NO: 4).

Example 3. 2′-FL Production in the Bioreactor

2′-FL can be produced in the bioreactor by any one of the host E. colistrains E214, E390 or E403, when transformed with any one of theplasmids pG171, pG175 or pG180. Growth of the transformed strain isperformed in a minimal medium in a bioreactor, 10 L working volume, withcontrol of dissolved oxygen, pH, lactose substrate, antifoam andnutrient levels. Minimal “FERM” medium is used in the bioreactor, whichis detailed below.

Ferm (10 liters): Minimal medium comprising:

-   -   40 g (NH₄)₂HPO₄    -   100 g KH₂PO₄    -   10 g MgSO₄.7H₂O    -   40 g NaOH    -   Trace elements:    -   1.3 g NTA    -   0.5 g FeSO₄.7H₂O    -   0.09 g MnCl₂.4H₂O    -   0.09 g ZnSO₄.7H₂O    -   0.01 g CoCl₂.6H₂O    -   0.01 g CuCl₂.2H₂O    -   0.02 g H₃BO₃    -   0.01 g Na₂MoO₄.2H₂O (pH 6.8)    -   Water to 10 liters    -   DF204 antifoam (0.1 ml/L)    -   150 g glycerol (initial batch growth), followed by fed batch        mode with a 90% glycerol-1% MgSO₄-1× trace elements feed, at        various rates for various times.

Production cell densities of A₆₀₀>100 are routinely achieved in thesebioreactor runs. Briefly, a small bacterial culture is grown overnightin “FERM”—in the absence of either antibiotic or exogenous thymidine.The overnight culture (@˜2 A₆₀₀) is used to inoculate a bioreactor (10 Lworking volume, containing “FERM”) to an initial cell density of ˜0.2A₆₀₀. Biomass is built up in batch mode at 30° C. until the glycerol isexhausted (A₆₀₀˜20), and then a fed batch phase is initiated utilizingglycerol as the limiting carbon source. At A₆₀₀˜ 30, 0.2 g/L tryptophanis added to induce α(1,2) fucosyltransferase synthesis. An initial bolusof lactose is also added at this time. 5 hr later, a continuous slowfeed of lactose is started in parallel to the glycerol feed. Theseconditions are continued for 48 hr (2′-FL production phase). At the endof this period, both the lactose and glycerol feeds are terminated, andthe residual glycerol and lactose are consumed over a final fermentationperiod, prior to harvest. 2′-FL accumulates in the spent fermentationmedium at concentrations as much as 30 times higher than in thecytoplasm. The specific yield in the spent medium varies between 10 and50 g/L, depending on precise growth and induction conditions. FIG. 18 isa TLC of culture medium samples removed from a bioreactor at varioustimes during a 2′-FL production run utilizing plasmid pG171 transformedinto strain E403. All of the input lactose was converted to product bythe end of the run, and product yield was approximately 25 g/L 2′-FL.

Example 4. 2′-Fucosyllactose Purification

2′-FL purification from E. coli fermentation broth is accomplishedthough five steps:

1. Clarification

Fermentation broth is harvested and cells removed by sedimentation in apreparative centrifuge at 6000×g for 30 min. Each bioreactor run yieldsabout 5-7 L of partially clarified supernatant. Clarified supernatantshave a brown/orange coloration attributed to a fraction of caramelizedsugars produced during the course of the fermentation, particularly byside-reactions promoted by the ammonium ions present in the fermentationmedium.

2. Product Capture on Coarse Carbon

A column packed with coarse carbon (Calgon 12×40 TR) of ˜1000 ml volume(dimension 5 cm diameter×60 cm length) is equilibrated with 1 columnvolume (CV) of water and loaded with clarified culture supernatant at aflow rate of 40 ml/min. This column has a total capacity of about 120 gof sugar (lactose). Following loading and sugar capture, the column iswashed with 1.5 CV of water, then eluted with 2.5 CV of 50% ethanol or25% isopropanol (lower concentrations of ethanol at this step (25-30%)may be sufficient for product elution). This solvent elution stepreleases about 95% of the total bound sugars on the column and a smallportion of the color bodies (caramels). In this first step capture ofthe maximal amount of sugar is the primary objective. Resolution ofcontaminants is not an objective. The column can be regenerated with a 5CV wash with water.

3. Evaporation

A volume of 2.5 L of ethanol or isopropanol eluate from the capturecolumn is rotary-evaporated at 56 C and a sugar syrup in water isgenerated (this typically is a yellow-brown color). Alternative methodsthat could be used for this step include lyophilization or spray-drying.

4. Flash Chromatography on Fine Carbon and Ion Exchange Media

A column (GE Healthcare HiScale50/40, 5×40 cm, max pressure 20 bar)connected to a Biotage Isolera One FLASH Chromatography System is packedwith 750 ml of a Darco Activated Carbon G60 (100-mesh): Celite 535(coarse) 1:1 mixture (both column packings obtained from Sigma). Thecolumn is equilibrated with 5 CV of water and loaded with sugar fromstep 3 (10-50 g, depending on the ratio of 2′-FL to contaminatinglactose), using either a celite loading cartridge or direct injection.The column is connected to an evaporative light scattering (ELSD)detector to detect peaks of eluting sugars during the chromatography. Afour-step gradient of isopropanol, ethanol or methanol is run in orderto separate 2′-FL from monosaccharides (if present), lactose and colorbodies. e.g., for B=ethanol: Step 1, 2.5 CV 0% B; Step 2, 4 CV 10% B(elutes monosaccharides and lactose contaminants); step 3, 4 CV 25% B(Elutes 2′-FL); step 4, 5 CV 50% B (elutes some of the color bodies andpartially regenerates the column). Additional column regeneration isachieved using methanol @ 50% and isopropanol @ 50%. Fractionscorresponding to sugar peaks are collected automatically in 120-mlbottles, pooled and directed to step 5. In certain purification runsfrom longer-than-normal fermentations, passage of the 2′-FL-containingfraction through anion-exchange and cation exchange columns can removeexcess protein/DNA/caramel body contaminants. Resins tested successfullyfor this purpose are Dowex 22 and Toyopearl Mono-Q, for the anionexchanger, and Dowex 88 for the cation exchanger. Mixed bed Dowex resinshave proved unsuitable as they tend to adsorb sugars at high affinityvia hydrophobic interactions. FIG. 19 illustrates the performance ofDarco G60:celite 1:1 in separating lactose from 2′-fucoyllactose whenused in Flash chromatography mode.

5. Evaporation/Lyophilization

3.0 L of 25% B solvent fractions is rotary-evaporated at 56 C until dry.Clumps of solid sugar are re-dissolved in a minimum amount of water, thesolution frozen, and then lyophilized. A white, crystalline, sweetpowder (2′-FL) is obtained at the end of the process. 2′-FL purityobtained lies between 95 and 99%.

Sugars are routinely analyzed for purity by spotting 1 μl aliquots onaluminum-backed silica G60 Thin Layer Chromatography plates (10×20 cm;Macherey-Nagel). A mixture of LDFT (Rf=0.18), 2′-FL (Rf=0.24), lactose(Rf=0.30), trehalose (Rf=0.32), acetyl-lactose (Rf=0.39) and fucose(Rf=0.48) (5 g/L concentration for each sugar) is run alongside asstandards. The plates are developed in a 50% butanol:25% acetic acid:25%water solvent until the front is within 1 cm from the top. Improvedsugar resolution can be obtained by performing two sequential runs,drying the plate between runs. Sugar spots are visualized by sprayingwith α-naphtol in a sulfuric acid-ethanol solution (2.4 g α-naphtol in83% (v/v) ethanol, 10.5% (v/v) sulfuric acid) and heating at 120 C for afew minutes. High molecular weight contaminants (DNA, protein, caramels)remain at the origin, or form smears with Rfs lower than LDFT.

Example 5. 3FL Production

Any one of E. coli host strains E214, E390 or E403, when transformedwith a plasmid expressing an α(1,3)fucosyltransferase capable of usinglactose as the sugar acceptor substrate, will produce the human milkoligosaccharide product, 3-fucosyllactose (3FL). FIG. 9 illustrates thepathways utilized in engineered strains of E. coli of this invention toachieve production of 3FL. For example, the plasmid pG176 (ColE1, thyA+,bla+, P_(L2)-futA, rcsA+) (SEQ ID NO: 2), is a derivative of pG175 inwhich the α(1,2) FT (wbsJ) sequence is replaced by the Helicobacterpylori futA gene (Dumon, C., Bosso, C., Utille, J. P., Heyraud, A. &Samain, E. Chembiochem 7, 359-365 (2006)). pG176 will direct theproduction of 3FL when transformed into any one of the host E. colistrains E214, E390 or E403. FIG. 11 shows a TLC analysis of 3FLproduction from E403 transformed with pG176. Additionally there areseveral other related bacterial-type α(1,3)-fucosyltransferasesidentified in Helicobacter pylori which could be used to directsynthesis of 3FL, e.g., “11639 FucTa” (Ge, Z., Chan, N. W., Palcic, M.M. & Taylor, D. E. J Biol Chem 272, 21357-21363 (1997); Martin, S. L.,Edbrooke, M. R., Hodgman, T. C., van den Eijnden, D. H. & Bird, M. I. JBiol Chem 272, 21349-21356 (1997)) and “UA948 FucTa” (Rasko, D. A.,Wang, G., Palcic, M. M. & Taylor, D. E. J Biol Chem 275, 4988-4994(2000)). In addition to α(1,3)-fucosyltransferases from H. pylori, anα(1,3)fucosyltransferase (Hh0072, sequence accession AAP76669) isolatedfrom Helicobacter hepaticus exhibits activity towards bothnon-sialylated and sialylated Type 2 oligosaccharide acceptor substrates(Zhang, L., Lau, K., Cheng, J., Yu, H., et al. Glycobiology (2010)).Furthermore, there are several additional bacterialα(1,3)-fucosyltransferases that may be used to make 3FL according to themethods of this invention. For example, close homologs of Hh0072 arefound in H. H. bilis (HRAG_01092 gene, sequence accession EEO24035), andin C. jejuni (C1336_000250319 gene, sequence accession EFC31050).

3FL biosynthesis is performed as described above for 2′-FL, either atsmall scale in culture tubes and culture flasks, or in a bioreactor (10L working volume) utilizing control of dissolved oxygen, pH, lactosesubstrate, antifoam and carbon:nitrogen balance. Cell densities ofA₆₀₀˜100 are reached in the bioreacter, and specific 3FL yields of up to3 g/L have been achieved. Approximately half of the 3FL produced isfound in the culture supernatant, and half inside the cells.Purification of 3FL from E. coli culture supernatants is achieved usingan almost identical procedure to that described above for 2′-FL. Theonly substantive difference being that 3FL elutes from carbon columns atlower alcohol concentrations than does 2′-FL.

Example 6. The Simultaneous Production of Human Milk Oligosaccharides2′-Fucosyllactose (2′-FL), 3-Fucosyllactose (3FL), andLactodifucohexaose (LDFT) in E. coli

E. coli strains E214, E390 and E403 accumulate cytoplasmic pools of bothlactose and GDP-fucose, as discussed above, and when transformed withplasmids expressing either an α(1,2) fucosyltransferase or an α(1,3)fucosyltransferase can synthesize the human milk oligosaccharides 2′-FLor 3FL respectively. The tetrasaccharide lactodifucotetrose (LDFT) isanother major fucosylated oligosaccharide found in human milk, andcontains both α(1,2)- and α(1,3)-linked fucose residues. pG177 (FIG. 10,SEQ ID NO: 3) is a derivative of pG175 in which the wbsJ gene isreplaced by a two gene operon comprising the Helicobacter pylori futAgene and the Helicobacter pylori futC gene (i.e., an operon containingboth an α(1,3)- and α(1,2)-fucosyltransferase). E. coli strains E214,E390 and E403 produce LDFT when transformed with plasmid pG177 andgrown, either in small scale or in the bioreactor, as described above.In FIG. 11 (lanes pG177), LDFT made in E. coli, directed by pG177, wasobserved on analysis of cell extracts by thin layer chromatography.

Example 7. 3′-SL Synthesis in the E. coli Cytoplasm

The first step in the production of 3′-sialyllactose (3′-SL) in E. coliis generation of a host background strain that accumulates cytoplasmicpools of both lactose and CMP-Neu5Ac (CMP-sialic acid). Accumulation ofcytoplasmic lactose is achieved through growth on lactose andinactivation of the endogenous E. coli β-galactosidase gene (lacZ),being careful to minimize polarity effects on lacY, the lac permease.This accumulation of a lactose pool has already been accomplished and isdescribed above in E. coli hosts engineered for 2′-FL, 3FL and LDFTproduction.

Specifically, a scheme to generate a cytoplasmic CMP-Neu5Ac pool,modified from methods known in the art, (e.g., Ringenberg, M.,Lichtensteiger, C. & Vimr, E. Glycobiology 11, 533-539 (2001); Fierfort,N. & Samain, E. J Biotechnol 134, 261-265 (2008)), is shown in FIG. 5.Under this scheme, the E. coli K12 sialic acid catabolic pathway isfirst ablated through introduction of null mutations in endogenous nanA(N-acetylneuraminate lyase) and nanK (N-acetylmannosamine kinase) genes.By “sialic acid catabolic pathway” is meant a sequence of reactions,usually controlled and catalyzed by enzymes, which results in thedegradation of sialic acid. An exemplary sialic acid catabolic pathwayin Escherichia coli is set forth in FIG. 5. In the sialic acid catabolicpathway in FIG. 5, sialic acid (Neu5Ac; N-acetylneuraminic acid) isdegraded by the enzymes NanA (N-acetylneuraminic acid lyase) and NanK(N-acetylmannosamine kinase). Other abbreviations for the sialic acidcatabolic pathway in FIG. 5 include: (nanT) sialic acid transporter;(AnanA) mutated N-acetylneuraminic acid lyase; (ΔnanK) mutatedN-acetylmannosamine kinase; (ManNAc-6-P)N-acetylmannosamine-6-phosphate; (GlcNAc-6-P)N-acetylglucosamine-6-phosphate; (GlcN-6-P) Glucosamine-6-phosphate;(Fruc-6-P) Fructose-6-phosphate; (neuA), CMP-N-acetylneuraminic acidsynthetase; (CMP-Neu5Ac) CMP-N-acetylneuraminic acid; and (neuB),N-acetylneuraminic acid synthase.

Next, since E. coli K12 lacks a de novo sialic acid synthesis pathway,sialic acid synthetic capability is introduced through the provision ofthree recombinant enzymes; a UDP-GlcNAc 2-epimerase (e.g., neuC), aNeu5Ac synthase (e.g., neuB) and a CMP-Neu5Ac synthetase (e.g., neuA).Equivalent genes from C. jejuni, E. coli K1, H. influenzae or from N.meningitides can be utilized (interchangeably) for this purpose.

The addition of sialic acid to the 3′ position of lactose to generate3′-sialyllactose is then achieved utilizing a bacterial-typeα(2,3)sialyltransferase, and numerous candidate genes have beendescribed, including those from N. meningitidis and N. gonorrhoeae(Gilbert, M., Watson, D. C., Cunningham, A. M., Jennings, M. P., et al.J Biol Chem 271, 28271-28276 (1996); Gilbert, M., Cunningham, A. M.,Watson, D. C., Martin, A., et al. Eur J Biochem 249, 187-194 (1997)).The Neisseria enzymes are already known to use lactose as an acceptorsugar. The recombinant N. meningitidis enzyme generates 3′-sialyllactosein engineered E. coli (Fierfort, N. & Samain, E. J Biotechnol 134,261-265 (2008)). FIG. 20 shows a TLC analysis of culture media takenfrom a culture of E. coli strain E547 (ampC::(Pr_(trpB)λcI⁺), P_(lacI)_(q) (ΔlacI-lacZ)₁₅₈lacY⁺, ΔacA, Δnan) and carrying plasmids expressingneuA,B,C and a bacterial-type α(2,3)sialyltransferase. The presence of3′-sialylactose (3′-SL) in the culture media is clearly seen.

Example 8. The Production of Human Milk Oligosaccharide3′-Sialyl-3-Fucosyllactose (3′-S3FL) in E. coli

Prior to the invention described herein, it was unpredictable that acombination of any particular fucosyltransferase gene and any particularsialyl-transferase gene in the same bacterial strain could produce3′-S3FL. Described below are results demonstrating that the combinationof a fucosyltransferase gene and a sialyl-transferase gene in the sameLac⁺ E. coli strain resulted in the production of 3′-S3FL. Theseunexpected results are likely due to the surprisingly relaxed substratespecificity of the particular fucosyltransferase and sialyl-transferaseenzymes utilzied.

Humans synthesize the sialyl-Lewis X epitope utilizing differentcombinations of six α(1,3)fucosyl- and six α(2,3)sialyl-transferasesencoded in the human genome (de Vries, T., Knegtel, R. M., Holmes, E. H.& Macher, B. A. Glycobiology 11, 119R-128R (2001); Taniguchi, A. CurrDrug Targets 9, 310-316 (2008)). These sugar transferases differ notonly in their tissue expression patterns, but also in their acceptorspecificities. For example, human myeloid-type α(1,3) fucosyltransferase(FUT IV) will fucosylate Type 2 (Galβ1->4Glc/GlcNAc) chain-basedacceptors, but only if they are non-sialylated. In contrast“plasma-type” α(1,3) fucosyltansferase (FUT VI) will utilize Type 2acceptors whether or not they are sialylated, and the promiscuous“Lewis” α(1,3/4) fucosyltransferase (FUT III), found in breast andkidney, will act on sialylated and non-sialylated Type 1(Galβ1->3GlcNAc) and Type 2 acceptors (Easton, E. W., Schiphorst, W. E.,van Drunen, E., van der Schoot, C. E. & van den Eijnden, D. H. Blood 81,2978-2986 (1993)). A similar situation exists for the family of human □α(2,3)sialyl-transferases, with different enzymes exhibiting majordifferences in acceptor specificity (Legaigneur, P., Breton, C., ElBattari, A., Guillemot, J. C., et al. J Biol Chem 276, 21608-21617(2001); Jeanneau, C., Chazalet, V., Augé, C., Soumpasis, D. M., et al. JBiol Chem 279, 13461-13468 (2004)). This diversity in acceptorspecificity highlights a key issue in the synthesis of3′-sialyl-3-fucosyllactose (3′-S3FL) in E. coli, i.e., to identify asuitable combination of fucosyl- and sialyl-transferases capable ofacting cooperatively to synthesize 3′-S3FL (utilizing lactose as theinitial acceptor sugar). However, since human and all other eukaryoticfucosyl- and sialyl-transferases are secreted proteins located in thelumen of the golgi, they are poorly suited for the task of 3′-S3FLbiosynthesis in the bacterial cytoplasm.

Several bacterial pathogens are known to incorporate fucosylated and/orsialylated sugars into their cell envelopes, typically for reasons ofhost mimicry and immune evasion. For example; both Neisseriameningitides and Campylobacter jejuni are able to incorporate sialicacid through 2,3-linkages to galactose moieties in their capsularlipooligosaccharide (LOS) (Tsai, C. M., Kao, G. & Zhu, P. I Infectionand Immunity 70, 407 (2002); Gilbert, M., Brisson, J. R., Karwaski, M.F., Michniewicz, J., et al. J Biol Chem 275, 3896-3906 (2000)), and somestrains of E. coli incorporate α(1,2) fucose groups intolipopolysaccharide (LPS) (Li, M., Liu, X. W., Shao, J., Shen, J., et al.Biochemistry 47, 378-387 (2008); Li, M., Shen, J., Liu, X., Shao, J., etal. Biochemistry 47, 11590-11597 (2008)). Certain strains ofHelicobacter pylori are able not only to incorporateα(2,3)-sialyl-groups, but also α(1,2)-, α(1,3)-, andα(1,4)-fucosyl-groups into LPS, and thus can display a broad range ofhuman Lewis-type epitopes on their cell surface (Moran, A. P. CarbohydrRes 343, 1952-1965 (2008)). Most bacterial sialyl- andfucosyl-transferases operate in the cytoplasm, i.e., they are bettersuited to the methods described herein than are eukaryoticgolgi-localized sugar transferases.

Strains of E. coli engineered to express the transferases describedabove accumulate a cytoplasmic pool of lactose, as well as an additionalpool of either the nucleotide sugar GDP-fucose, or the nucleotide sugarCMP-Neu5Ac (CMP-sialic acid). Addition of these sugars to the lactoseacceptor is performed in these engineered hosts using candidaterecombinant α(1,3)-fucosyl- or α(2,3)-sialyl-transferases, generating3-fucosyllactose and 3′-sialyllactose respectively. Finally, the twosynthetic capabilities are combined into a single E. coli strain toproduce 3′-S3FL.

An E. coli strain that accumulates cytoplasmic pools of both lactose andGDP-fucose has been developed. This strain, when transformed with aplasmid over-expressing an α(1,2)fucosyltransferase, produces2′-fucosyllactose (2′-FL) at levels of ˜10-50 g/L of bacterial culturemedium. A substitution of the α(1,2) fucosyltransferase in this hostwith an appropriate α(1,3) fucosyltransferase leads to the production of3-fucosyllactose (3FL). The bacterial α(1,3) fucosyltransferase thenworks in conjunction with a bacterial α(2,3)sialyltransferase to makethe desired product, 3′-S3FL.

An α(1,3)fucosyltransferase (Hh0072) isolated from Helicobacterhepaticus exhibits activity towards both non-sialylated and sialylatedType 2 oligosaccharide acceptor substrates (Zhang, L., Lau, K., Cheng,J., Yu, H., et al. Glycobiology (2010)). This enzyme is cloned,expressed, and evaluated to measure utilization of a lactose acceptorand to evaluate production of 3FL in the context of the currentGDP-fucose-producing E. coli host. Hh0072 is also tested in concert withvarious bacterial α(2,3)sialyltransferases for its competence in 3′-S3FLsynthesis. As alternatives to Hh0072, there are two characterizedhomologous bacterial-type 3-fucosyltransferases identified inHelicobacter pylori, “11639 FucTa” (Ge, Z., Chan, N. W., Palcic, M. M. &Taylor, D. E. J Biol Chem 272, 21357-21363 (1997); Martin, S. L.,Edbrooke, M. R., Hodgman, T. C., van den Eijnden, D. H. & Bird, M. I. JBiol Chem 272, 21349-21356 (1997)) and “UA948 FucTa” (Rasko, D. A.,Wang, G., Palcic, M. M. & Taylor, D. E. J Biol Chem 275, 4988-4994(2000)). These two paralogs exhibit differing acceptor specificities,“11639 FucTa” utilizes only Type 2 acceptors and is a strictα(1,3)-fucosyltransferase, whereas “UA948 FucTa” has relaxed acceptorspecificity (utilizing both Type1 and Type 2 acceptors) and is able togenerate both α(1,3)- and α(1,4)-fucosyl linkages. The precise molecularbasis of this difference in specificity was determined (Ma, B., Lau, L.H., Palcic, M. M., Hazes, B. & Taylor, D. E. J Biol Chem 280,36848-36856 (2005)), and characterization of several additionalα(1,3)-fucosyltransferase paralogs from a variety of additional H.pylori strains revealed significant strain-to-strain acceptorspecificity diversity.

In addition to the enzymes from H. pylori and H. hepaticus, otherbacterial α(1,3)-fucosyltransferases are optionally used. For example,close homologs of Hh0072 are found in H. bilis (HRAG_01092 gene,sequence accession EEO24035), and in C. jejuni (C1336_000250319 gene,sequence accession EFC31050).

Described below is 3′-S3FL synthesis in E. coli. The first step towardsthis is to combine into a single E. coli strain the 3-fucosyllactosesynthetic ability, outlined above, with the ability to make3′-sialyllactose, also outlined above. All of the chromosomal geneticmodifications discussed above are introduced into a new host strain,which will then simultaneously accumulate cytoplasmic pools of the 3specific precursors; lactose, GDP-fucose and CMP-Neu5Ac. This “combined”strain background is then used to host simultaneous production of anα(1,3)fucosyltransferase with an α(2,3)sialyltransferase, with geneexpression driven either off two compatible multicopy plasmids or withboth enzyme genes positioned on the same plasmid as an artificialoperon. Acceptor specificities for some of the bacterialα(1,3)fucosyltransferases and α(2,3)sialyltransferases, particularlywith respect to fucosylation of 3′-sialyllactose and sialylation of3-fucosyllactose and different combinations of α(1,3)fucosyltransferaseand α(2,3)sialyltransferase enzymes are evaluated. Production levels andratios of 3′-SL, 3FL and 3′-S3FL are monitored, e.g., by TLC, withconfirmation of identity by NMR and accurate quantitation either bycalibrated mass spectrometry utilizing specific ion monitoring, or bycapillary electrophoresis (Bao, Y., Zhu, L. & Newburg, D. S.Simultaneous quantification of sialyloligosaccharides from human milk bycapillary electrophoresis. Anal Biochem 370, 206-214 (2007)).

The sequences corresponding to the SEQ ID NOs described herein areprovided below.

The sequence of PG175 is set forth below (SEQ ID NO: 1):

TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCTCCTCAACCTGTATATTCGTAAACCACGCCCAATGGGAGCTGTCTCAGGTTTGTTCCTGATTGGTTACGGCGCGTTTCGCATCATTGTTGAGTTTTTCCGCCAGCCCGACGCGCAGTTTACCGGTGCCTGGGTGCAGTACATCAGCATGGGGCAAATTCTTTCCATCCCGATGATTGTCGCGGGTGTGATCATGATGGTCTGGGCATATCGTCGCAGCCCACAGCAACACGTTTCCTGAGGAACCATGAAACAGTATTTAGAACTGATGCAAAAAGTGCTCGACGAAGGCACACAGAAAAACGACCGTACCGGAACCGGAACGCTTTCCATTTTTGGTCATCAGATGCGTTTTAACCTGCAAGATGGATTCCCGCTGGTGACAACTAAACGTTGCCACCTGCGTTCCATCATCCATGAACTGCTGTGGTTTCTGCAGGGCGACACTAACATTGCTTATCTACACGAAAACAATGTCACCATCTGGGACGAATGGGCCGATGAAAACGGCGACCTCGGGCCAGTGTATGGTAAACAGTGGCGCGCCTGGCCAACGCCAGATGGTCGTCATATTGACCAGATCACTACGGTACTGAACCAGCTGAAAAACGACCCGGATTCGCGCCGCATTATTGTTTCAGCGTGGAACGTAGGCGAACTGGATAAAATGGCGCTGGCACCGTGCCATGCATTCTTCCAGTTCTATGTGGCAGACGGCAAACTCTCTTGCCAGCTTTATCAGCGCTCCTGTGACGTCTTCCTCGGCCTGCCGTTCAACATTGCCAGCTACGCGTTATTGGTGCATATGATGGCGCAGCAGTGCGATCTGGAAGTGGGTGATTTTGTCTGGACCGGTGGCGACACGCATCTGTACAGCAACCATATGGATCAAACTCATCTGCAATTAAGCCGCGAACCGCGTCCGCTGCCGAAGTTGATTATCAAACGTAAACCCGAATCCATCTTCGACTACCGTTTCGAAGACTTTGAGATTGAAGGCTACGATCCGCATCCGGGCATTAAAGCGCCGGTGGCTATCTAATTACGAAACATCCTGCCAGAGCCGACGCCAGTGTGCGTCGGTTTTTTTACCCTCCGTTAAATTCTTCGAGACGCCTTCCCGAAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTTCTTTAATGAAGCAGGGCATCAGGACGGTATCTTTGTGGAGAAAGCAGAGTAATCTTATTCAGCCTGACTGGTGGGAAACCACCAGTCAGAATGTGTTAGCGCATGTTGACAAAAATACCATTAGTCACATTATCCGTCAGTCGGACGACATGGTAGATAACCTGTTTATTATGCGTTTTGATCTTACGTTTAATATTACCTTTATGCGATGAAACGGTCTTGGCTTTGATATTCATTTGGTCAGAGATTTGAATGGTTCCCTGACCTGCCATCCACATTCGCAACATACTCGATTCGGTTCGGCTCAATGATAACGTCGGCATATTTAAAAACGAGGTTATCGTTGTCTCTTTTTTCAGAATATCGCCAAGGATATCGTCGAGAGATTCCGGTTTAATCGATTTAGAACTGATCAATAAATTTTTTCTGACCAATAGATATTCATCAAAATGAACATTGGCAATTGCCATAAAAACGATAAATAACGTATTGGGATGTTGATTAATGATGAGCTTGATACGCTGACTGTTAGAAGCATCGTGGATGAAACAGTCCTCATTAATAAACACCACTGAAGGGCGCTGTGAATCACAAGCTATGGCAAGGTCATCAACGGTTTCAATGTCGTTGATTTCTCTTTTTTTAACCCCTCTACTCAACAGATACCCGGTTAAACCTAGTCGGGTGTAACTACATAAATCCATAATAATCGTTGACATGGCATACCCTCACTCAATGCGTAACGATAATTCCCCTTACCTGAATATTTCATCATGACTAAACGGAACAACATGGGTCACCTAATGCGCCACTCTCGCGATTTTTCAGGCGGACTTACTATCCCGTAAAGTGTTGTATAATTTGCCTGGAATTGTCTTAAAGTAAAGTAAATGTTGCGATATGTGAGTGAGCTTAAAACAAATATTTCGCTGCAGGAGTATCCTGGAAGATGTTCGTAGAAGCTTACTGCTCACAAGAAAAAAGGCACGTCATCTGACGTGCCTTTTTTATTTGTACTACCCTGTACGATTACTGCAGCTCGAGTTATTATAATTTTACCCACGATTCGGGAATAATATCATGTTTAATATCTTTCTTAAACCATTTACTCGGAGCAATTACTGTTTTATTTTTATTTTCATTTAACCAAGCAGCCCACCAACTGAAAGAACTATTTGAAATTATATTATTTTTACATTTACTCATAAGCAGCATATCTAATTCAACATGATAAGCATCACCTTGAACAAAACATATTTGATTATTAAAAAATATATTTTCCCTGCACCACTTTATATCATCAGAAAAAATGAAGAGAAGGGTTTTTTTATTAATAACACCTTTATTCATCAAATAATCAATGGCACGTTCAAAATATTTTTCACTACATGTGCCATGAGTTTCATTTGCTATTTTACTGGAAACATAATCACCTCTTCTAATATGTAATGAACAAGTATCATTTTCTTTAATTAAATTAAGCAATTCATTTTGATAACTATTAAACTTGGTTTTAGGTTGAAATTCCTTTATCAACTCATGCCTAAATTCCTTAAAATATTTTTCAGTTTGAAAATAACCGACGATTTTTTTATTTATACTTTTGGTATCAATATCTGGATCATACTCTAAACTTTTCTCAACGTAATGCTTTCTGAACATTCCTTTTTTCATGAAATGTGGGATTTTTTCGGAAAATAAGTATTTTTCAAATGGCCATGCTTTTTTTACAAATTCTGAACTACAAGATAATTCAACTAATCTTAATGGATGAGTTTTATATTTTACTGCATCAGATATATCAACAGTCAAATTTTGATGAGTTCTTTTTGCAATAGCAAATGCAGTTGCATACTGAAACATTTGATTACCAAGACCACCAATAATTTTAACTTCCATATGTATATCTCCTTCTTCTAGAATTCTAAAAATTGATTGAATGTATGCAAATAAATGCATACACCATAGGTGTGGTTTAATTTGATGCCCTTTTTCAGGGCTGGAATGTGTAAGAGCGGGGTTATTTATGCTGTTGTTTTTTTGTTACTCGGGAAGGGCTTTACCTCTTCCGCATAAACGCTTCCATCAGCGTTTATAGTTAAAAAAATCTTTCGGAACTGGTTTTGCGCTTACCCCAACCAACAGGGGATTTGCTGCTTTCCATTGAGCCTGTTTCTCTGCGCGACGTTCGCGGCGGCGTGTTTGTGCATCCATCTGGATTCTCCTGTCAGTTAGCTTTGGTGGTGTGTGGCAGTTGTAGTCCTGAACGAAAACCCCCCGCGATTGGCACATTGGCAGCTAATCCGGAATCGCACTTACGGCCAATGCTTCGTTTCGTATCACACACCCCAAAGCCTTCTGCTTTGAATGCTGCCCTTCTTCAGGGCTTAATTTTTAAGAGCGTCACCTTCATGGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCACCGCCAGTGGTATTTATGTCAACACCGCCAGAGATAATTTATCACCGCAGATGGTTATCTGTATGTTTTTTATATGAATTTATTTTTTGCAGGGGGGCATTGTTTGGTAGGTGAGAGATCAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC

The sequence of pG176 is set forth below (SEQ ID NO: 2):

TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATGAAACAGTATTTAGAACTGATGCAAAAAGTGCTCGACGAAGGCACACAGAAAAACGACCGTACCGGAACCGGAACGCTTTCCATTTTTGGTCATCAGATGCGTTTTAACCTGCAAGATGGATTCCCGCTGGTGACAACTAAACGTTGCCACCTGCGTTCCATCATCCATGAACTGCTGTGGTTTCTGCAGGGCGACACTAACATTGCTTATCTACACGAAAACAATGTCACCATCTGGGACGAATGGGCCGATGAAAACGGCGACCTCGGGCCAGTGTATGGTAAACAGTGGCGCGCCTGGCCAACGCCAGATGGTCGTCATATTGACCAGATCACTACGGTACTGAACCAGCTGAAAAACGACCCGGATTCGCGCCGCATTATTGTTTCAGCGTGGAACGTAGGCGAACTGGATAAAATGGCGCTGGCACCGTGCCATGCATTCTTCCAGTTCTATGTGGCAGACGGCAAACTCTCTTGCCAGCTTTATCAGCGCTCCTGTGACGTCTTCCTCGGCCTGCCGTTCAACATTGCCAGCTACGCGTTATTGGTGCATATGATGGCGCAGCAGTGCGATCTGGAAGTGGGTGATTTTGTCTGGACCGGTGGCGACACGCATCTGTACAGCAACCATATGGATCAAACTCATCTGCAATTAAGCCGCGAACCGCGTCCGCTGCCGAAGTTGATTATCAAACGTAAACCCGAATCCATCTTCGACTACCGTTTCGAAGACTTTGAGATTGAAGGCTACGATCCGCATCCGGGCATTAAAGCGCCGGTGGCTATCTAAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTTCTTTAATGAAGCAGGGCATCAGGACGGTATCTTTGTGGAGAAAGCAGAGTAATCTTATTCAGCCTGACTGGTGGGAAACCACCAGTCAGAATGTGTTAGCGCATGTTGACAAAAATACCATTAGTCACATTATCCGTCAGTCGGACGACATGGTAGATAACCTGTTTATTATGCGTTTTGATCTTACGTTTAATATTACCTTTATGCGATGAAACGGTCTTGGCTTTGATATTCATTTGGTCAGAGATTTGAATGGTTCCCTGACCTGCCATCCACATTCGCAACATACTCGATTCGGTTCGGCTCAATGATAACGTCGGCATATTTAAAAACGAGGTTATCGTTGTCTCTTTTTTCAGAATATCGCCAAGGATATCGTCGAGAGATTCCGGTTTAATCGATTTAGAACTGATCAATAAATTTTTTCTGACCAATAGATATTCATCAAAATGAACATTGGCAATTGCCATAAAAACGATAAATAACGTATTGGGATGTTGATTAATGATGAGCTTGATACGCTGACTGTTAGAAGCATCGTGGATGAAACAGTCCTCATTAATAAACACCACTGAAGGGCGCTGTGAATCACAAGCTATGGCAAGGTCATCAACGGTTTCAATGTCGTTGATTTCTCTTTTTTTAACCCCTCTACTCAACAGATACCCGGTTAAACCTAGTCGGGTGTAACTACATAAATCCATAATAATCGTTGACATGGCATACCCTCACTCAATGCGTAACGATAATTCCCCTTACCTGAATATTTCATCATGACTAAACGGAACAACATGGGTCACCTAATGCGCCACTCTCGCGATTTTTCAGGCGGACTTACTATCCCGTAAAGTGTTGTATAATTTGCCTGGAATTGTCTTAAAGTAAAGTAAATGTTGCGATATGTGAGTGAGCTTAAAACAAATATTTCGCTGCAGGAGTATCCTGGAAGATGTTCGTAGAAGCTTACTGCTCACAAGAAAAAAGGCACGTCATCTGACGTGCCTTTTTTATTTGTACTACCCTGTACGATTACTGCAGCTCGAGTTAATTCAAATCTTCTTCAGAAATCAATTTTTGTTCCAAACCCAATTTTTTAACCAACTTTCTCACCGCGCGCAACAAAGGCAAGGATTTTTGATAAGCTTTGCGATAGATTTTAAAAGTGGTGTTTTGAGAGAGTTCTAATAAAGGCGAAGCGTTTTGTAAAAGCCGGTCATAATTAACCCTCAAATCATCATAATTAACCCTCAAATCATCAATGGATACTAACGGCTTATGCAGATCGTACTCCCACATGAAAGATGTTGAGAATTTGTGATAAATCGTATCGTTTTCTAAAATCGTTTTAAAAAAATCTAGGATTTTTTTAAAACTCAAATCTTGGTAAAAGTAAGCTTTCCCATCAAGGGTGTTTAAAGGGTTTTCATAGAGCATGTCTAAATAAGCGTTTGGGTGCGTGTGCAGGTATTTGATATAATCAATCGCTTCATCAAAGTTGTTGAAATCATGCACATTCACAAAACTTTTAGGGTTAAAATCTTTCGCCACGCTGGGACTCCCCCAATAAATAGGAATGGTATGGCTAAAATACGCATCAAGGATTTTTTCGGTTACATAGCCATAACCTTGCGAGTTTTCAAAACAGAGATTGAACTTGTATTGGCTTAAAAACTCGCTTTTGTTTCCAACCTTATAGCCTAAAGTGTTTCTCACACTTCCTCCCCCAGTAACTGGCTCTATGGAATTTAGAGCGTCATAAAAAGCGTTCCTCATAGGAGCGTTAGCGTTGCTCGCTACAAAACTGGCAAACCCTCTTTTTAAAAGATCGCTCTCATCATTCACTACTGCGCACAAATTAGGGTGGTTTTCTTTAAAATGATGAGAGGGTTTTTTTAAAGCATAAAGGCTGTTGTCTTTGAGTTTGTAGGGCGCAGTGGTGTCATTAACAAGCTCGGCTTTATAGTGCAAATGGGCATAATACAAAGGCATTCTCAAATAACGATCATTAAAATCCAATTCATCAAAGCCTATGGCGTAATCAAAGAGGTTGAAATTAGGTGATTCGTTTTCACCGGTGTAAAACACTCGTTTAGTGTTTTGATAAGATAAAATCTTTCTAGCCGCTCCAAGAGGATTGCTAAAAACTAGATCTGAAAATTCATTGGGGTTTTGGTGGAGGGTGATTGCGTAGCGTTGGCTTAGGATAAAATAAAGAACGCTCTTTTTAAATTCTTTAATTTCTTCATCTCCCCACCAATTCGCCACAGCGATTTTTAGGGGGGGGGGGGGAGATTTAGAGGCCATTTTTTCAATGGAAGCGCTTTCTATAAAGGCGTCTAATAGGGGTTGGAACATATGTATATCTCCTTCTTGAATTCTAAAAATTGATTGAATGTATGCAAATAAATGCATACACCATAGGTGTGGTTTAATTTGATGCCCTTTTTCAGGGCTGGAATGTGTAAGAGCGGGGTTATTTATGCTGTTGTTTTTTTGTTACTCGGGAAGGGCTTTACCTCTTCCGCATAAACGCTTCCATCAGCGTTTATAGTTAAAAAAATCTTTCGGAACTGGTTTTGCGCTTACCCCAACCAACAGGGGATTTGCTGCTTTCCATTGAGCCTGTTTCTCTGCGCGACGTTCGCGGCGGCGTGTTTGTGCATCCATCTGGATTCTCCTGTCAGTTAGCTTTGGTGGTGTGTGGCAGTTGTAGTCCTGAACGAAAACCCCCCGCGATTGGCACATTGGCAGCTAATCCGGAATCGCACTTACGGCCAATGCTTCGTTTCGTATCACACACCCCAAAGCCTTCTGCTTTGAATGCTGCCCTTCTTCAGGGCTTAATTTTTAAGAGCGTCACCTTCATGGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCACCGCCAGTGGTATTTATGTCAACACCGCCAGAGATAATTTATCACCGCAGATGGTTATCTGTATGTTTTTTATATGAATTTATTTTTTGCAGGGGGGCATTGTTTGGTAGGTGAGAGATCAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC

The sequence of pG177 is set forth below (SEQ ID NO: 3):

TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATGAAACAGTATTTAGAACTGATGCAAAAAGTGCTCGACGAAGGCACACAGAAAAACGACCGTACCGGAACCGGAACGCTTTCCATTTTTGGTCATCAGATGCGTTTTAACCTGCAAGATGGATTCCCGCTGGTGACAACTAAACGTTGCCACCTGCGTTCCATCATCCATGAACTGCTGTGGTTTCTGCAGGGCGACACTAACATTGCTTATCTACACGAAAACAATGTCACCATCTGGGACGAATGGGCCGATGAAAACGGCGACCTCGGGCCAGTGTATGGTAAACAGTGGCGCGCCTGGCCAACGCCAGATGGTCGTCATATTGACCAGATCACTACGGTACTGAACCAGCTGAAAAACGACCCGGATTCGCGCCGCATTATTGTTTCAGCGTGGAACGTAGGCGAACTGGATAAAATGGCGCTGGCACCGTGCCATGCATTCTTCCAGTTCTATGTGGCAGACGGCAAACTCTCTTGCCAGCTTTATCAGCGCTCCTGTGACGTCTTCCTCGGCCTGCCGTTCAACATTGCCAGCTACGCGTTATTGGTGCATATGATGGCGCAGCAGTGCGATCTGGAAGTGGGTGATTTTGTCTGGACCGGTGGCGACACGCATCTGTACAGCAACCATATGGATCAAACTCATCTGCAATTAAGCCGCGAACCGCGTCCGCTGCCGAAGTTGATTATCAAACGTAAACCCGAATCCATCTTCGACTACCGTTTCGAAGACTTTGAGATTGAAGGCTACGATCCGCATCCGGGCATTAAAGCGCCGGTGGCTATCTAAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTTCTTTAATGAAGCAGGGCATCAGGACGGTATCTTTGTGGAGAAAGCAGAGTAATCTTATTCAGCCTGACTGGTGGGAAACCACCAGTCAGAATGTGTTAGCGCATGTTGACAAAAATACCATTAGTCACATTATCCGTCAGTCGGACGACATGGTAGATAACCTGTTTATTATGCGTTTTGATCTTACGTTTAATATTACCTTTATGCGATGAAACGGTCTTGGCTTTGATATTCATTTGGTCAGAGATTTGAATGGTTCCCTGACCTGCCATCCACATTCGCAACATACTCGATTCGGTTCGGCTCAATGATAACGTCGGCATATTTAAAAACGAGGTTATCGTTGTCTCTTTTTTCAGAATATCGCCAAGGATATCGTCGAGAGATTCCGGTTTAATCGATTTAGAACTGATCAATAAATTTTTTCTGACCAATAGATATTCATCAAAATGAACATTGGCAATTGCCATAAAAACGATAAATAACGTATTGGGATGTTGATTAATGATGAGCTTGATACGCTGACTGTTAGAAGCATCGTGGATGAAACAGTCCTCATTAATAAACACCACTGAAGGGCGCTGTGAATCACAAGCTATGGCAAGGTCATCAACGGTTTCAATGTCGTTGATTTCTCTTTTTTTAACCCCTCTACTCAACAGATACCCGGTTAAACCTAGTCGGGTGTAACTACATAAATCCATAATAATCGTTGACATGGCATACCCTCACTCAATGCGTAACGATAATTCCCCTTACCTGAATATTTCATCATGACTAAACGGAACAACATGGGTCACCTAATGCGCCACTCTCGCGATTTTTCAGGCGGACTTACTATCCCGTAAAGTGTTGTATAATTTGCCTGGAATTGTCTTAAAGTAAAGTAAATGTTGCGATATGTGAGTGAGCTTAAAACAAATATTTCGCTGCAGGAGTATCCTGGAAGATGTTCGTAGAAGCTTACTGCTCACAAGAAAAAAGGCACGTCATCTGACGTGCCTTTTTTATTTGTACTACCCTGTACGATTACTGCAGCTCGAGTTAATTCAAATCTTCTTCAGAAATCAATTTTTGTTCAGCGTTATACTTTTGGGATTTTACCTCAAAATGGGATTCTATTTTCACCCACTCCTTACAAAGGATATTCTCATGCCCAAAAAGCCAGTGTTTGGGGCCAATAATGATTTTTTCTGGATTTTCTATCAAATAGGCCGCCCACCAGCTATAAGTGCTATTAGCGATAATGCCATGCTGACAAGATTGCATGAGCAGCATGTCCCAATACGCCTCTTCTTCTTTATCCCTAGTGGTCATGTCCATAAAAGGGTAGCCAAGATCAAGATTTTGCGTGAATTCTAAGTCTTCGCAAAACACAAAAAGCTCCATGTTTGGCACGCGCTTTGCCATATACTCAAGCGCCTTTTTTTGATAGTCAATACCAAGCTGACAGCCAATCCCCACATAATCCCCTCTTCTTATATGCACAAACACGCTGTTTTTAGCGGCTAAAATCAAAGAAAGCTTGCACTGATATTCTTCCTCTTTTTTATTATTATTCTTATTATTTTCGGGTGGTGGTGGTAGAGTGAAGGTTTGCTTGATTAAAGGGGATATAGCATCAAAGTATCGTGGATCTTGGAAATAGCCAAAAAAATAAGTCAAGCGGCTTGGCTTTAGCAATTTAGGCTCGTATTCAAAAACGATTTCTTGACTCACCCTATCAAATCCCATGCATTTGAGCGCGTCTCTTACTAGCTTGGGGAGGTGTTGCATTTTAGCTATAGCGATTTCTTTCGCGCTCGCATAGGGCAAATCAATAGGGAAAAGTTCTAATTGCATTTTCCTATCGCTCCAATCAAAAGAAGTGATATCTAACAGCACAGGCGTATTAGAGTGTTTTTGCAAACTTTTAGCGAAAGCGTATTGAAACATTTGATTCCCAAGCCCTCCGCAAATTTGCACCACCTTAAAAGCCATATGTATATCTCCTTCTTGCTCGAGTTAATTCAAATCTTCTTCAGAAATCAATTTTTGTTCCAAACCCAATTTTTTAACCAACTTTCTCACCGCGCGCAACAAAGGCAAGGATTTTTGATAAGCTTTGCGATAGATTTTAAAAGTGGTGTTTTGAGAGAGTTCTAATAAAGGCGAAGCGTTTTGTAAAAGCCGGTCATAATTAACCCTCAAATCATCATAATTAACCCTCAAATCATCAATGGATACTAACGGCTTATGCAGATCGTACTCCCACATGAAAGATGTTGAGAATTTGTGATAAATCGTATCGTTTTCTAAAATCGTTTTAAAAAAATCTAGGATTTTTTTAAAACTCAAATCTTGGTAAAAGTAAGCTTTCCCATCAAGGGTGTTTAAAGGGTTTTCATAGAGCATGTCTAAATAAGCGTTTGGGTGCGTGTGCAGGTATTTGATATAATCAATCGCTTCATCAAAGTTGTTGAAATCATGCACATTCACAAAACTTTTAGGGTTAAAATCTTTCGCCACGCTGGGACTCCCCCAATAAATAGGAATGGTATGGCTAAAATACGCATCAAGGATTTTTTCGGTTACATAGCCATAACCTTGCGAGTTTTCAAAACAGAGATTGAACTTGTATTGGCTTAAAAACTCGCTTTTGTTTCCAACCTTATAGCCTAAAGTGTTTCTCACACTTCCTCCCCCAGTAACTGGCTCTATGGAATTTAGAGCGTCATAAAAAGCGTTCCTCATAGGAGCGTTAGCGTTGCTCGCTACAAAACTGGCAAACCCTCTTTTTAAAAGATCGCTCTCATCATTCACTACTGCGCACAAATTAGGGTGGTTTTCTTTAAAATGATGAGAGGGTTTTTTTAAAGCATAAAGGCTGTTGTCTTTGAGTTTGTAGGGCGCAGTGGTGTCATTAACAAGCTCGGCTTTATAGTGCAAATGGGCATAATACAAAGGCATTCTCAAATAACGATCATTAAAATCCAATTCATCAAAGCCTATGGCGTAATCAAAGAGGTTGAAATTAGGTGATTCGTTTTCACCGGTGTAAAACACTCGTTTAGTGTTTTGATAAGATAAAATCTTTCTAGCCGCTCCAAGAGGATTGCTAAAAACTAGATCTGAAAATTCATTGGGGTTTTGGTGGAGGGTGATTGCGTAGCGTTGGCTTAGGATAAAATAAAGAACGCTCTTTTTAAATTCTTTAATTTCTTCATCTCCCCACCAATTCGCCACAGCGATTTTTAGGGGGGGGGGGGGAGATTTAGAGGCCATTTTTTCAATGGAAGCGCTTTCTATAAAGGCGTCTAATAGGGGTTGGAACATATGTATATCTCCTTCTTGAATTCTAAAAATTGATTGAATGTATGCAAATAAATGCATACACCATAGGTGTGGTTTAATTTGATGCCCTTTTTCAGGGCTGGAATGTGTAAGAGCGGGGTTATTTATGCTGTTGTTTTTTTGTTACTCGGGAAGGGCTTTACCTCTTCCGCATAAACGCTTCCATCAGCGTTTATAGTTAAAAAAATCTTTCGGAACTGGTTTTGCGCTTACCCCAACCAACAGGGGATTTGCTGCTTTCCATTGAGCCTGTTTCTCTGCGCGACGTTCGCGGCGGCGTGTTTGTGCATCCATCTGGATTCTCCTGTCAGTTAGCTTTGGTGGTGTGTGGCAGTTGTAGTCCTGAACGAAAACCCCCCGCGATTGGCACATTGGCAGCTAATCCGGAATCGCACTTACGGCCAATGCTTCGTTTCGTATCACACACCCCAAAGCCTTCTGCTTTGAATGCTGCCCTTCTTCAGGGCTTAATTTTTAAGAGCGTCACCTTCATGGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCACCGCCAGTGGTATTTATGTCAACACCGCCAGAGATAATTTATCACCGCAGATGGTTATCTGTATGTTTTTTATATGAATTTATTTTTTGCAGGGGGGCATTGTTTGGTAGGTGAGAGATCAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC

The sequence of Bacteroides fragilis NCTC 9343 wcfW CDS DNA is set forthe below (SEQ ID NO: 4):

ATGATTGTATCATCTTTGCGAGGAGGATTGGGGAATCAAATGTTTATTTACGCTATGGTGAAGGCCATGGCATTAAGAAACAATGTACCATTCGCTTTTAATTTGACTACTGATTTTGCAAATGATGAAGTTTATAAAAGGAAACTTTTATTATCATATTTTGCATTAGACTTGCCTGAAAATAAAAAATTAACATTTGATTTTTCATATGGGAATTATTATAGAAGGCTAAGTCGTAATTTAGGTTGTCATATACTTCATCCATCATATCGTTATATTTGCGAAGAGCGCCCTCCCCACTTTGAATCAAGGTTAATTAGTTCTAAGATTACAAATGCTTTTCTGGAAGGATATTGGCAGTCAGAAAAATATTTTCTTGATTATAAACAAGAGATAAAAGAGGACTTTGTAATACAAAAAAAATTAGAATACACATCGTATTTGGAATTGGAAGAAATAAAATTGCTAGATAAGAATGCCATAATGATTGGGGTTAGACGGTATCAGGAAAGTGATGTAGCTCCTGGTGGAGTGTTAGAAGATGATTACTATAAATGTGCTATGGATATTATGGCATCAAAAGTTACTTCTCCTGTTTTCTTTTGTTTTTCACAAGATTTAGAATGGGTTGAAAAACATCTAGCGGGAAAATATCCTGTTCGTTTGATAAGTAAAAAGGAGGATGATAGTGGTACTATAGATGATATGTTTCTAATGATGCATTTTCGTAATTATATAATATCGAATAGCTCTTTTTACTGGTGGGGAGCATGGCTTTCGAAATATGATGATAAGCTGGTGATTGCTCCAGGTAATTTTATAAATAAGGATTCTGTACCAGAATCTTGGT TTAAATTGAATGTAAGATAA

The sequence of pG171 is set forth below (SEQ ID NO: 5):

TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCTCCTCAACCTGTATATTCGTAAACCACGCCCAATGGGAGCTGTCTCAGGTTTGTTCCTGATTGGTTACGGCGCGTTTCGCATCATTGTTGAGTTTTTCCGCCAGCCCGACGCGCAGTTTACCGGTGCCTGGGTGCAGTACATCAGCATGGGGCAAATTCTTTCCATCCCGATGATTGTCGCGGGTGTGATCATGATGGTCTGGGCATATCGTCGCAGCCCACAGCAACACGTTTCCTGAGGAACCATGAAACAGTATTTAGAACTGATGCAAAAAGTGCTCGACGAAGGCACACAGAAAAACGACCGTACCGGAACCGGAACGCTTTCCATTTTTGGTCATCAGATGCGTTTTAACCTGCAAGATGGATTCCCGCTGGTGACAACTAAACGTTGCCACCTGCGTTCCATCATCCATGAACTGCTGTGGTTTCTGCAGGGCGACACTAACATTGCTTATCTACACGAAAACAATGTCACCATCTGGGACGAATGGGCCGATGAAAACGGCGACCTCGGGCCAGTGTATGGTAAACAGTGGCGCGCCTGGCCAACGCCAGATGGTCGTCATATTGACCAGATCACTACGGTACTGAACCAGCTGAAAAACGACCCGGATTCGCGCCGCATTATTGTTTCAGCGTGGAACGTAGGCGAACTGGATAAAATGGCGCTGGCACCGTGCCATGCATTCTTCCAGTTCTATGTGGCAGACGGCAAACTCTCTTGCCAGCTTTATCAGCGCTCCTGTGACGTCTTCCTCGGCCTGCCGTTCAACATTGCCAGCTACGCGTTATTGGTGCATATGATGGCGCAGCAGTGCGATCTGGAAGTGGGTGATTTTGTCTGGACCGGTGGCGACACGCATCTGTACAGCAACCATATGGATCAAACTCATCTGCAATTAAGCCGCGAACCGCGTCCGCTGCCGAAGTTGATTATCAAACGTAAACCCGAATCCATCTTCGACTACCGTTTCGAAGACTTTGAGATTGAAGGCTACGATCCGCATCCGGGCATTAAAGCGCCGGTGGCTATCTAATTACGAAACATCCTGCCAGAGCCGACGCCAGTGTGCGTCGGTTTTTTTACCCTCCGTTAAATTCTTCGAGACGCCTTCCCGAAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTTCTTTAATGAAGCAGGGCATCAGGACGGTATCTTTGTGGAGAAAGCAGAGTAATCTTATTCAGCCTGACTGGTGGGAAACCACCAGTCAGAATGTGTTAGCGCATGTTGACAAAAATACCATTAGTCACATTATCCGTCAGTCGGACGACATGGTAGATAACCTGTTTATTATGCGTTTTGATCTTACGTTTAATATTACCTTTATGCGATGAAACGGTCTTGGCTTTGATATTCATTTGGTCAGAGATTTGAATGGTTCCCTGACCTGCCATCCACATTCGCAACATACTCGATTCGGTTCGGCTCAATGATAACGTCGGCATATTTAAAAACGAGGTTATCGTTGTCTCTTTTTTCAGAATATCGCCAAGGATATCGTCGAGAGATTCCGGTTTAATCGATTTAGAACTGATCAATAAATTTTTTCTGACCAATAGATATTCATCAAAATGAACATTGGCAATTGCCATAAAAACGATAAATAACGTATTGGGATGTTGATTAATGATGAGCTTGATACGCTGACTGTTAGAAGCATCGTGGATGAAACAGTCCTCATTAATAAACACCACTGAAGGGCGCTGTGAATCACAAGCTATGGCAAGGTCATCAACGGTTTCAATGTCGTTGATTTCTCTTTTTTTAACCCCTCTACTCAACAGATACCCGGTTAAACCTAGTCGGGTGTAACTACATAAATCCATAATAATCGTTGACATGGCATACCCTCACTCAATGCGTAACGATAATTCCCCTTACCTGAATATTTCATCATGACTAAACGGAACAACATGGGTCACCTAATGCGCCACTCTCGCGATTTTTCAGGCGGACTTACTATCCCGTAAAGTGTTGTATAATTTGCCTGGAATTGTCTTAAAGTAAAGTAAATGTTGCGATATGTGAGTGAGCTTAAAACAAATATTTCGCTGCAGGAGTATCCTGGAAGATGTTCGTAGAAGCTTACTGCTCACAAGAAAAAAGGCACGTCATCTGACGTGCCTTTTTTATTTGTACTACCCTGTACGATTACTGCAGCTCGAGTTTAATTCAAATCTTCTTCAGAAATCAATTTTTGTTCAGCGTTATACTTTTGGGATTTTACCTCAAAATGGGATTCTATTTTCACCCACTCCTTACAAAGGATATTCTCATGCCCAAAAAGCCAGTGTTTGGGGCCAATAATGATTTTTTCTGGATTTTCTATCAAATAGGCCGCCCACCAGCTATAAGTGCTATTAGCGATAATGCCATGCTGACAAGATTGCATGAGCAGCATGTCCCAATACGCCTCTTCTTCTTTATCCCTAGTGGTCATGTCCATAAAAGGGTAGCCAAGATCAAGATTTTGCGTGAATTCTAAGTCTTCGCAAAACACAAAAAGCTCCATGTTTGGCACGCGCTTTGCCATATACTCAAGCGCCTTTTTTTGATAGTCAATACCAAGCTGACAGCCAATCCCCACATAATCCCCTCTTCTTATATGCACAAACACGCTGTTTTTAGCGGCTAAAATCAAAGAAAGCTTGCACTGATATTCTTCCTCTTTTTTATTATTATTCTTATTATTTTCGGGTGGTGGTGGTAGAGTGAAGGTTTGCTTGATTAAAGGGGATATAGCATCAAAGTATCGTGGATCTTGGAAATAGCCAAAAAAATAAGTCAAGCGGCTTGGCTTTAGCAATTTAGGCTCGTATTCAAAAACGATTTCTTGACTCACCCTATCAAATCCCATGCATTTGAGCGCGTCTCTTACTAGCTTGGGGAGGTGTTGCATTTTAGCTATAGCGATTTCTTTCGCGCTCGCATAGGGCAAATCAATAGGGAAAAGTTCTAATTGCATTTTCCTATCGCTCCAATCAAAAGAAGTGATATCTAACAGCACAGGCGTATTAGAGTGTTTTTGCAAACTTTTAGCGAAAGCGTATTGAAACATTTGATTCCCAAGCCCTCCGCAAATTTGCACCACCTTAAAAGCCATATGTATATCTCCTTCTTGAATTCTAAAAATTGATTGAATGTATGCAAATAAATGCATACACCATAGGTGTGGTTTAATTTGATGCCCTTTTTCAGGGCTGGAATGTGTAAGAGCGGGGTTATTTATGCTGTTGTTTTTTTGTTACTCGGGAAGGGCTTTACCTCTTCCGCATAAACGCTTCCATCAGCGTTTATAGTTAAAAAAATCTTTCGGAACTGGTTTTGCGCTTACCCCAACCAACAGGGGATTTGCTGCTTTCCATTGAGCCTGTTTCTCTGCGCGACGTTCGCGGCGGCGTGTTTGTGCATCCATCTGGATTCTCCTGTCAGTTAGCTTTGGTGGTGTGTGGCAGTTGTAGTCCTGAACGAAAACCCCCCGCGATTGGCACATTGGCAGCTAATCCGGAATCGCACTTACGGCCAATGCTTCGTTTCGTATCACACACCCCAAAGCCTTCTGCTTTGAATGCTGCCCTTCTTCAGGGCTTAATTTTTAAGAGCGTCACCTTCATGGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCACCGCCAGTGGTATTTATGTCAACACCGCCAGAGATAATTTATCACCGCAGATGGTTATCTGTATGTTTTTTATATGAATTTATTTTTTGCAGGGGGGCATTGTTTGGTAGGTGAGAGATCAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC

The sequence of pG180 is set forth below (SEQ ID NO: 6):

TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCTCCTCAACCTGTATATTCGTAAACCACGCCCAATGGGAGCTGTCTCAGGTTTGTTCCTGATTGGTTACGGCGCGTTTCGCATCATTGTTGAGTTTTTCCGCCAGCCCGACGCGCAGTTTACCGGTGCCTGGGTGCAGTACATCAGCATGGGGCAAATTCTTTCCATCCCGATGATTGTCGCGGGTGTGATCATGATGGTCTGGGCATATCGTCGCAGCCCACAGCAACACGTTTCCTGAGGAACCATGAAACAGTATTTAGAACTGATGCAAAAAGTGCTCGACGAAGGCACACAGAAAAACGACCGTACCGGAACCGGAACGCTTTCCATTTTTGGTCATCAGATGCGTTTTAACCTGCAAGATGGATTCCCGCTGGTGACAACTAAACGTTGCCACCTGCGTTCCATCATCCATGAACTGCTGTGGTTTCTGCAGGGCGACACTAACATTGCTTATCTACACGAAAACAATGTCACCATCTGGGACGAATGGGCCGATGAAAACGGCGACCTCGGGCCAGTGTATGGTAAACAGTGGCGCGCCTGGCCAACGCCAGATGGTCGTCATATTGACCAGATCACTACGGTACTGAACCAGCTGAAAAACGACCCGGATTCGCGCCGCATTATTGTTTCAGCGTGGAACGTAGGCGAACTGGATAAAATGGCGCTGGCACCGTGCCATGCATTCTTCCAGTTCTATGTGGCAGACGGCAAACTCTCTTGCCAGCTTTATCAGCGCTCCTGTGACGTCTTCCTCGGCCTGCCGTTCAACATTGCCAGCTACGCGTTATTGGTGCATATGATGGCGCAGCAGTGCGATCTGGAAGTGGGTGATTTTGTCTGGACCGGTGGCGACACGCATCTGTACAGCAACCATATGGATCAAACTCATCTGCAATTAAGCCGCGAACCGCGTCCGCTGCCGAAGTTGATTATCAAACGTAAACCCGAATCCATCTTCGACTACCGTTTCGAAGACTTTGAGATTGAAGGCTACGATCCGCATCCGGGCATTAAAGCGCCGGTGGCTATCTAATTACGAAACATCCTGCCAGAGCCGACGCCAGTGTGCGTCGGTTTTTTTACCCTCCGTTAAATTCTTCGAGACGCCTTCCCGAAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTTCTTTAATGAAGCAGGGCATCAGGACGGTATCTTTGTGGAGAAAGCAGAGTAATCTTATTCAGCCTGACTGGTGGGAAACCACCAGTCAGAATGTGTTAGCGCATGTTGACAAAAATACCATTAGTCACATTATCCGTCAGTCGGACGACATGGTAGATAACCTGTTTATTATGCGTTTTGATCTTACGTTTAATATTACCTTTATGCGATGAAACGGTCTTGGCTTTGATATTCATTTGGTCAGAGATTTGAATGGTTCCCTGACCTGCCATCCACATTCGCAACATACTCGATTCGGTTCGGCTCAATGATAACGTCGGCATATTTAAAAACGAGGTTATCGTTGTCTCTTTTTTCAGAATATCGCCAAGGATATCGTCGAGAGATTCCGGTTTAATCGATTTAGAACTGATCAATAAATTTTTTCTGACCAATAGATATTCATCAAAATGAACATTGGCAATTGCCATAAAAACGATAAATAACGTATTGGGATGTTGATTAATGATGAGCTTGATACGCTGACTGTTAGAAGCATCGTGGATGAAACAGTCCTCATTAATAAACACCACTGAAGGGCGCTGTGAATCACAAGCTATGGCAAGGTCATCAACGGTTTCAATGTCGTTGATTTCTCTTTTTTTAACCCCTCTACTCAACAGATACCCGGTTAAACCTAGTCGGGTGTAACTACATAAATCCATAATAATCGTTGACATGGCATACCCTCACTCAATGCGTAACGATAATTCCCCTTACCTGAATATTTCATCATGACTAAACGGAACAACATGGGTCACCTAATGCGCCACTCTCGCGATTTTTCAGGCGGACTTACTATCCCGTAAAGTGTTGTATAATTTGCCTGGAATTGTCTTAAAGTAAAGTAAATGTTGCGATATGTGAGTGAGCTTAAAACAAATATTTCGCTGCAGGAGTATCCTGGAAGATGTTCGTAGAAGCTTACTGCTCACAAGAAAAAAGGCACGTCATCTGACGTGCCTTTTTTATTTGTACTACCCTGTACGATTACTGCAGCTCGAGTTTAATTCAAATCTTCTTCAGAAATCAATTTTTGTTCTCTTACATTCAATTTAAACCAAGATTCTGGTACAGAATCCTTATTTATAAAATTACCTGGAGCAATCACCAGCTTATCATCATATTTCGAAAGCCATGCTCCCCACCAGTAAAAAGAGCTATTCGATATTATATAATTACGAAAATGCATCATTAGAAACATATCATCTATAGTACCACTATCATCCTCCTTTTTACTTATCAAACGAACAGGATATTTTCCCGCTAGATGTTTTTCAACCCATTCTAAATCTTGTGAAAAACAAAAGAAAACAGGAGAAGTAACTTTTGATGCCATAATATCCATAGCACATTTATAGTAATCATCTTCTAACACTCCACCAGGAGCTACATCACTTTCCTGATACCGTCTAACCCCAATCATTATGGCATTCTTATCTAGCAATTTTATTTCTTCCAATTCCAAATACGATGTGTATTCTAATTTTTTTTGTATTACAAAGTCCTCTTTTATCTCTTGTTTATAATCAAGAAAATATTTTTCTGACTGCCAATATCCTTCCAGAAAAGCATTTGTAATCTTAGAACTAATTAACCTTGATTCAAAGTGGGGAGGGCGCTCTTCGCAAATATAACGATATGATGGATGAAGTATATGACAACCTAAATTACGACTTAGCCTTCTATAATAATTCCCATATGAAAAATCAAATGTTAATTTTTTATTTTCAGGCAAGTCTAATGCAAAATATGATAATAAAAGTTTCCTTTTATAAACTTCATCATTTGCAAAATCAGTAGTCAAATTAAAAGCGAATGGTACATTGTTTCTTAATGCCATGGCCTTCACCATAGCGTAAATAAACATTTGATTCCCCAATCCTCCTCGCAAAGATGATACAATCATATGTATATCTCCTTCTTGTCTAGAATTCTAAAAATTGATTGAATGTATGCAAATAAATGCATACACCATAGGTGTGGTTTAATTTGATGCCCTTTTTCAGGGCTGGAATGTGTAAGAGCGGGGTTATTTATGCTGTTGTTTTTTTGTTACTCGGGAAGGGCTTTACCTCTTCCGCATAAACGCTTCCATCAGCGTTTATAGTTAAAAAAATCTTTCGGAACTGGTTTTGCGCTTACCCCAACCAACAGGGGATTTGCTGCTTTCCATTGAGCCTGTTTCTCTGCGCGACGTTCGCGGCGGCGTGTTTGTGCATCCATCTGGATTCTCCTGTCAGTTAGCTTTGGTGGTGTGTGGCAGTTGTAGTCCTGAACGAAAACCCCCCGCGATTGGCACATTGGCAGCTAATCCGGAATCGCACTTACGGCCAATGCTTCGTTTCGTATCACACACCCCAAAGCCTTCTGCTTTGAATGCTGCCCTTCTTCAGGGCTTAATTTTTAAGAGCGTCACCTTCATGGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCACCGCCAGTGGTATTTATGTCAACACCGCCAGAGATAATTTATCACCGCAGATGGTTATCTGTATGTTTTTTATATGAATTTATTTTTTGCAGGGGGGCATTGTTTGGTAGGTGAGAGATCAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC

The sequence of W3110 deltalon::Kan::lacZwithRBS Escherichia coli str.K-12 substr. W3110 is set forth below (SEQ ID NO: 7):

GTCCATGGAAGACGTCGAAAAAGTGGTTATCGACGAGTCGGTAATTGATGGTCAAAGCAAACCGTTGCTGATTTATGGCAAGCCGGAAGCGCAACAGGCATCTGGTGAATAATTAACCATTCCCATACAATTAGTTAACCAAAAAGGGGGGATTTTATCTCCCCTTTAATTTTTCCTCTATTCTCGGCGTTGAATGTGGGGGAAACATCCCCATATACTGACGTACATGTTAATAGATGGCGTGAAGCACAGTCGTGTCATCTGATTACCTGGCGGAAATTAAACTAAGAGAGAGCTCTATGATTCCGGGGATCCGTCGACCTGCAGTTCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCAGAGCGCTTTTGAAGCTCACGCTGCCGCAAGCACTCAGGGCGCAAGGGCTGCTAAAGGAAGCGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTAATAAGGGGATCTTGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCGAAGCAGCTCCAGCCTACATAAAGCGGCCGCTTATTTTTGACACCAGACCAACTGGTAATGGTAGCGACCGGCGCTCAGCTGGAATTCCGCCGATACTGACGGGCTCCAGGAGTCGTCGCCACCAATCCCCATATGGAAACCGTCGATATTCAGCCATGTGCCTTCTTCCGCGTGCAGCAGATGGCGATGGCTGGTTTCCATCAGTTGCTGTTGACTGTAGCGGCTGATGTTGAACTGGAAGTCGCCGCGCCACTGGTGTGGGCCATAATTCAATTCGCGCGTCCCGCAGCGCAGACCGTTTTCGCTCGGGAAGACGTACGGGGTATACATGTCTGACAATGGCAGATCCCAGCGGTCAAAACAGGCGGCAGTAAGGCGGTCGGGATAGTTTTCTTGCGGCCCTAATCCGAGCCAGTTTACCCGCTCTGCTACCTGCGCCAGCTGGCAGTTCAGGCCAATCCGCGCCGGATGCGGTGTATCGCTCGCCACTTCAACATCAACGGTAATCGCCATTTGACCACTACCATCAATCCGGTAGGTTTTCCGGCTGATAAATAAGGTTTTCCCCTGATGCTGCCACGCGTGAGCGGTCGTAATCAGCACCGCATCAGCAAGTGTATCTGCCGTGCACTGCAACAACGCTGCTTCGGCCTGGTAATGGCCCGCCGCCTTCCAGCGTTCGACCCAGGCGTTAGGGTCAATGCGGGTCGCTTCACTTACGCCAATGTCGTTATCCAGCGGTGCACGGGTGAACTGATCGCGCAGCGGCGTCAGCAGTTGTTTTTTATCGCCAATCCACATCTGTGAAAGAAAGCCTGACTGGCGGTTAAATTGCCAACGCTTATTACCCAGCTCGATGCAAAAATCCATTTCGCTGGTGGTCAGATGCGGGATGGCGTGGGACGCGGCGGGGAGCGTCACACTGAGGTTTTCCGCCAGACGCCACTGCTGCCAGGCGCTGATGTGCCCGGCTTCTGACCATGCGGTCGCGTTCGGTTGCACTACGCGTACTGTGAGCCAGAGTTGCCCGGCGCTCTCCGGCTGCGGTAGTTCAGGCAGTTCAATCAACTGTTTACCTTGTGGAGCGACATCCAGAGGCACTTCACCGCTTGCCAGCGGCTTACCATCCAGCGCCACCATCCAGTGCAGGAGCTCGTTATCGCTATGACGGAACAGGTATTCGCTGGTCACTTCGATGGTTTGCCCGGATAAACGGAACTGGAAAAACTGCTGCTGGTGTTTTGCTTCCGTCAGCGCTGGATGCGGCGTGCGGTCGGCAAAGACCAGACCGTTCATACAGAACTGGCGATCGTTCGGCGTATCGCCAAAATCACCGCCGTAAGCCGACCACGGGTTGCCGTTTTCATCATATTTAATCAGCGACTGATCCACCCAGTCCCAGACGAAGCCGCCCTGTAAACGGGGATACTGACGAAACGCCTGCCAGTATTTAGCGAAACCGCCAAGACTGTTACCCATCGCGTGGGCGTATTCGCAAAGGATCAGCGGGCGCGTCTCTCCAGGTAGCGAAAGCCATTTTTTGATGGACCATTTCGGCACAGCCGGGAAGGGCTGGTCTTCATCCACGCGCGCGTACATCGGGCAAATAATATCGGTGGCCGTGGTGTCGGCTCCGCCGCCTTCATACTGCACCGGGCGGGAAGGATCGACAGATTTGATCCAGCGATACAGCGCGTCGTGATTAGCGCCGTGGCCTGATTCATTCCCCAGCGACCAGATGATCACACTCGGGTGATTACGATCGCGCTGCACCATTCGCGTTACGCGTTCGCTCATCGCCGGTAGCCAGCGCGGATCATCGGTCAGACGATTCATTGGCACCATGCCGTGGGTTTCAATATTGGCTTCATCCACCACATACAGGCCGTAGCGGTCGCACAGCGTGTACCACAGCGGATGGTTCGGATAATGCGAACAGCGCACGGCGTTAAAGTTGTTCTGCTTCATCAGCAGGATATCCTGCACCATCGTCTGCTCATCCATGACCTGACCATGCAGAGGATGATGCTCGTGACGGTTAACGCCTCGAATCAGCAACGGCTTGCCGTTCAGCAGCAGCAGACCATTTTCAATCCGCACCTCGCGGAAACCGACATCGCAGGCTTCTGCTTCAATCAGCGTGCCGTCGGCGGTGTGCAGTTCAACCACCGCACGATAGAGATTCGGGATTTCGGCGCTCCACAGTTTCGGGTTTTCGACGTTCAGACGTAGTGTGACGCGATCGGCATAACCACCACGCTCATCGATAATTTCACCGCCGAAAGGCGCGGTGCCGCTGGCGACCTGCGTTTCACCCTGCCATAAAGAAACTGTTACCCGTAGGTAGTCACGCAACTCGCCGCACATCTGAACTTCAGCCTCCAGTACAGCGCGGCTGAAATCATCATTAAAGCGAGTGGCAACATGGAAATCGCTGATTTGTGTAGTCGGTTTATGCAGCAACGAGACGTCACGGAAAATGCCGCTCATCCGCCACATATCCTGATCTTCCAGATAACTGCCGTCACTCCAGCGCAGCACCATCACCGCGAGGCGGTTTTCTCCGGCGCGTAAAAATGCGCTCAGGTCAAATTCAGACGGCAAACGACTGTCCTGGCCGTAACCGACCCAGCGCCCGTTGCACCACAGATGAAACGCCGAGTTAACGCCATCAAAAATAATTCGCGTCTGGCCTTCCTGTAGCCAGCTTTCATCAACATTAAATGTGAGCGAGTAACAACCCGTCGGATTCTCCGTGGGAACAAACGGCGGATTGACCGTAATGGGATAGGTCACGTTGGTGTAGATGGGCGCATCGTAACCGTGCATCTGCCAGTTTGAGGGGACGACGACAGTATCGGCCTCAGGAAGATCGCACTCCAGCCAGCTTTCCGGCACCGCTTCTGGTGCCGGAAACCAGGCAAAGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATCCGTAATCATGGTCATAGTAGGTTTCCTCAGGTTGTGACTGCAAAATAGTGACCTCGCGCAAAATGCACTAATAAAAACAGGGCTGGCAGGCTAATTCGGGCTTGCCAGCCTTTTTTTGTCTCGCTAAGTTAGATGGCGGATCGGGCTTGCCCTTATTAAGGGGTGTTGTAAGGGGATGGCTGGCCTGATATAACTGCTGCGCGTTCGTACCTTGAAGGATTCAAGTGCGATATAAATTATAAAGAGGAAGAGAAGAGTGAATAAATCTCAATTGATCGACAAGATTGCTGCAGGGGCTGATATCTCTAAAGCTGCGGCTGGCCGTGCGTTAGATGCTATTATTGCTTCCGTAACTGAATCTCTGAAAGAAGG

The sequence of pG186 is set forth below (SEQ ID NO: 8):

TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCTCCTCAACCTGTATATTCGTAAACCACGCCCAATGGGAGCTGTCTCAGGTTTGTTCCTGATTGGTTACGGCGCGTTTCGCATCATTGTTGAGTTTTTCCGCCAGCCCGACGCGCAGTTTACCGGTGCCTGGGTGCAGTACATCAGCATGGGGCAAATTCTTTCCATCCCGATGATTGTCGCGGGTGTGATCATGATGGTCTGGGCATATCGTCGCAGCCCACAGCAACACGTTTCCTGAGGAACCATGAAACAGTATTTAGAACTGATGCAAAAAGTGCTCGACGAAGGCACACAGAAAAACGACCGTACCGGAACCGGAACGCTTTCCATTTTTGGTCATCAGATGCGTTTTAACCTGCAAGATGGATTCCCGCTGGTGACAACTAAACGTTGCCACCTGCGTTCCATCATCCATGAACTGCTGTGGTTTCTGCAGGGCGACACTAACATTGCTTATCTACACGAAAACAATGTCACCATCTGGGACGAATGGGCCGATGAAAACGGCGACCTCGGGCCAGTGTATGGTAAACAGTGGCGCGCCTGGCCAACGCCAGATGGTCGTCATATTGACCAGATCACTACGGTACTGAACCAGCTGAAAAACGACCCGGATTCGCGCCGCATTATTGTTTCAGCGTGGAACGTAGGCGAACTGGATAAAATGGCGCTGGCACCGTGCCATGCATTCTTCCAGTTCTATGTGGCAGACGGCAAACTCTCTTGCCAGCTTTATCAGCGCTCCTGTGACGTCTTCCTCGGCCTGCCGTTCAACATTGCCAGCTACGCGTTATTGGTGCATATGATGGCGCAGCAGTGCGATCTGGAAGTGGGTGATTTTGTCTGGACCGGTGGCGACACGCATCTGTACAGCAACCATATGGATCAAACTCATCTGCAATTAAGCCGCGAACCGCGTCCGCTGCCGAAGTTGATTATCAAACGTAAACCCGAATCCATCTTCGACTACCGTTTCGAAGACTTTGAGATTGAAGGCTACGATCCGCATCCGGGCATTAAAGCGCCGGTGGCTATCTAATTACGAAACATCCTGCCAGAGCCGACGCCAGTGTGCGTCGGTTTTTTTACCCTCCGTTAAATTCTTCGAGACGCCTTCCCGAAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTTCTTTAATGAAGCAGGGCATCAGGACGGTATCTTTGTGGAGAAAGCAGAGTAATCTTATTCAGCCTGACTGGTGGGAAACCACCAGTCAGAATGTGTTAGCGCATGTTGACAAAAATACCATTAGTCACATTATCCGTCAGTCGGACGACATGGTAGATAACCTGTTTATTATGCGTTTTGATCTTACGTTTAATATTACCTTTATGCGATGAAACGGTCTTGGCTTTGATATTCATTTGGTCAGAGATTTGAATGGTTCCCTGACCTGCCATCCACATTCGCAACATACTCGATTCGGTTCGGCTCAATGATAACGTCGGCATATTTAAAAACGAGGTTATCGTTGTCTCTTTTTTCAGAATATCGCCAAGGATATCGTCGAGAGATTCCGGTTTAATCGATTTAGAACTGATCAATAAATTTTTTCTGACCAATAGATATTCATCAAAATGAACATTGGCAATTGCCATAAAAACGATAAATAACGTATTGGGATGTTGATTAATGATGAGCTTGATACGCTGACTGTTAGAAGCATCGTGGATGAAACAGTCCTCATTAATAAACACCACTGAAGGGCGCTGTGAATCACAAGCTATGGCAAGGTCATCAACGGTTTCAATGTCGTTGATTTCTCTTTTTTTAACCCCTCTACTCAACAGATACCCGGTTAAACCTAGTCGGGTGTAACTACATAAATCCATAATAATCGTTGACATGGCATACCCTCACTCAATGCGTAACGATAATTCCCCTTACCTGAATATTTCATCATGACTAAACGGAACAACATGGGTCACCTAATGCGCCACTCTCGCGATTTTTCAGGCGGACTTACTATCCCGTAAAGTGTTGTATAATTTGCCTGGAATTGTCTTAAAGTAAAGTAAATGTTGCGATATGTGAGTGAGCTTAAAACAAATATTTCGCTGCAGGAGTATCCTGGAAGATGTTCGTAGAAGCTTACTGCTCACAAGAAAAAAGGCACGTCATCTGACGTGCCTTTTTTATTTGTACTACCCTGTACGATTACTGCAGCTCGAGTTAGTCTTTATCTGCCGGACTTAAGGTCACTGAAGAGAGATAATTCAGCAGGGCGATATCGTTCTCGACACCCAGCTTCATCATCGCAGATTTCTTCTGGCTACTGATGGTTTTAATACTGCGGTTCAGCTTTTTAGCGATCTCGGTCACCAGGAAGCCTTCCGCAAACAGGCGCAGAACTTCACTCTCTTTTGGCGAGAGACGCTTGTCACCGTAACCACCAGCACTGATTTTTTCCAACAGGCGAGAAACGCTTTCCGGGGTAAATTTCTTCCCTTTCTGCAGCGCGGCGAGAGCTTTCGGCAGATCGGTCGGTGCACCTTGTTTCAGCACGATCCCTTCGATATCCAGATCCAATACCGCACTAAGAATCGCCGGGTTGTTGTTCATAGTCAGAACAATGATCGACAGGCTTGGGAAATGGCGCTTGATGTACTTGATTAAGGTAATGCCATCGCCGTACTTATCGCCAGGCATGGAGAGATCGGTAATCAACACATGCGCATCCAGTTTCGGCAGGTTGTTGATCAGTGCTGTAGAGTCTTCAAATTCGCCGACAACATTCACCCACTCAATTTGCTCAAGTGATTTGCGAATACCGAACAAGACTATCGGATGGTCATCGGCAATAATTACGTTCATATTGTTCATTGTATATCTCCTTCTTCTCGAGTTTAATTCAAATCTTCTTCAGAAATCAATTTTTGTTCAGCGTTATACTTTTGGGATTTTACCTCAAAATGGGATTCTATTTTCACCCACTCCTTACAAAGGATATTCTCATGCCCAAAAAGCCAGTGTTTGGGGCCAATAATGATTTTTTCTGGATTTTCTATCAAATAGGCCGCCCACCAGCTATAAGTGCTATTAGCGATAATGCCATGCTGACAAGATTGCATGAGCAGCATGTCCCAATACGCCTCTTCTTCTTTATCCCTAGTGGTCATGTCCATAAAAGGGTAGCCAAGATCAAGATTTTGCGTGAATTCTAAGTCTTCGCAAAACACAAAAAGCTCCATGTTTGGCACGCGCTTTGCCATATACTCAAGCGCCTTTTTTTGATAGTCAATACCAAGCTGACAGCCAATCCCCACATAATCCCCTCTTCTTATATGCACAAACACGCTGTTTTTAGCGGCTAAAATCAAAGAAAGCTTGCACTGATATTCTTCCTCTTTTTTATTATTATTCTTATTATTTTCGGGTGGTGGTGGTAGAGTGAAGGTTTGCTTGATTAAAGGGGATATAGCATCAAAGTATCGTGGATCTTGGAAATAGCCAAAAAAATAAGTCAAGCGGCTTGGCTTTAGCAATTTAGGCTCGTATTCAAAAACGATTTCTTGACTCACCCTATCAAATCCCATGCATTTGAGCGCGTCTCTTACTAGCTTGGGGAGGTGTTGCATTTTAGCTATAGCGATTTCTTTCGCGCTCGCATAGGGCAAATCAATAGGGAAAAGTTCTAATTGCATTTTCCTATCGCTCCAATCAAAAGAAGTGATATCTAACAGCACAGGCGTATTAGAGTGTTTTTGCAAACTTTTAGCGAAAGCGTATTGAAACATTTGATTCCCAAGCCCTCCGCAAATTTGCACCACCTTAAAAGCCATATGTATATCTCCTTCTTGAATTCTAAAAATTGATTGAATGTATGCAAATAAATGCATACACCATAGGTGTGGTTTAATTTGATGCCCTTTTTCAGGGCTGGAATGTGTAAGAGCGGGGTTATTTATGCTGTTGTTTTTTTGTTACTCGGGAAGGGCTTTACCTCTTCCGCATAAACGCTTCCATCAGCGTTTATAGTTAAAAAAATCTTTCGGAACTGGTTTTGCGCTTACCCCAACCAACAGGGGATTTGCTGCTTTCCATTGAGCCTGTTTCTCTGCGCGACGTTCGCGGCGGCGTGTTTGTGCATCCATCTGGATTCTCCTGTCAGTTAGCTTTGGTGGTGTGTGGCAGTTGTAGTCCTGAACGAAAACCCCCCGCGATTGGCACATTGGCAGCTAATCCGGAATCGCACTTACGGCCAATGCTTCGTTTCGTATCACACACCCCAAAGCCTTCTGCTTTGAATGCTGCCCTTCTTCAGGGCTTAATTTTTAAGAGCGTCACCTTCATGGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCACCGCCAGTGGTATTTATGTCAACACCGCCAGAGATAATTTATCACCGCAGATGGTTATCTGTATGTTTTTTATATGAATTTATTTTTTGCAGGGGGGCATTGTTTGGTAGGTGAGAGATCAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference.

Genbank and NCBI submissions indicated by accession number cited hereinare hereby incorporated by reference. All other published references,documents, manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A method for producing a fucosylatedoligosaccharide in an E.coli bacterium, comprising (a) providing anE.coli bacterium comprising a β-galactosidase activity between 0.05 and5 units and a mutation in a lacA gene, wherein said E.coli bacteriumfurther comprises an exogenous lactose-accepting fucosyltransferasegene; (b) culturing said E.coli bacterium in the presence of lactose;and (c) retrieving a fucosylated oligosaccharide from said E.colibacterium or from a culture supernatant of said E.coli bacterium,wherein a functional β-galactosidase gene is inserted into an endogenousgene of said E. coli bacterium.
 2. The method of claim 1, wherein saidmutation prevents the formation of intracellular acetyl-lactose.
 3. Themethod of claim 1, wherein said E.coli bacterium further comprises adefective colanic acid synthesis pathway.
 4. The method of claim 1,wherein said defective colanic acid synthesis pathway comprises amutation in a colanic acid synthesis gene.
 5. The method of claim 4,wherein said colanic acid synthesis gene comprises a wcaJ, wzxC, wcaD,wza, wzb, or wzc gene.
 6. The method of claim 1, further comprising aninactivating mutation in a Ion gene.
 7. The method of claim 6, whereinsaid bacterium comprises a functional promoter-less wild-type E. colilacZ⁺ gene inserted into said Ion gene.
 8. The method of claim 1,wherein said bacterium further comprises an exogenous E. coli rcsA or E.coli rcsB gene.
 9. The method of claim 1, wherein said exogenouslactose-accepting fucosyltransferase gene comprises a Bacteroidesfragilis wcfW gene or a Helicobacter pylori 26695 futA gene.
 10. Themethod of claim 1, wherein said bacterium comprises both an exogenousfucosyltransferase gene encoding α(1,2) fucosyltransferase and anexogenous fucosyltransferase gene encoding α(1,3) fucosyltransferase.11. The method of claim 1, wherein said bacterium comprises anendogenous lacY gene.
 12. The method of claim 1, wherein said bacteriumcomprises a lactose permease gene.
 13. The method of claim 12, whereinsaid lactose permease gene comprises an exogenous lactose permease gene.14. The method of claim 1, wherein said E.coli bacterium comprises adeletion of an endogenous β-galactosidase gene.
 15. The method of claim1, wherein said bacterium accumulates an increased cytoplasmic lactoselevel, wherein the increased intracellular lactose level is at least 10%more than the level in a corresponding wild type bacterium.
 16. Themethod of claim 1, wherein said fucosylated oligosaccharide comprises2′-fucosyllactose, 3-fucosyllactose, or lactodifucotetraose.
 17. Themethod of claim 1, wherein culturing said bacterium in step (b) does notcomprise an antibiotic.
 18. The method of claim 1, wherein saidbacterium comprises a plasmid expression vector.
 19. The method of claim1, wherein said bacterium comprises an exogenous fucosyltransferase geneencoding α(1,2) fucosyltransferase or an exogenous fucosyltransferasegene encoding α(1,3) fucosyltransferase.
 20. The method of claim 1,wherein said exogenous lactose-accepting fucosyltransferase genecomprises an α(1,3) fucosyltransferase gene from Helicobacter pylori ,H. hepaticus, H. bilis, Campylobacter jejuni, or a species ofBacteroides.
 21. The method of claim 1, wherein said exogenouslactose-accepting fucosyltransferase gene comprises a E. coli strainO128:B12 wbsJ gene, a Helicobacter pylori 26695 futC gene, a H.hepaticus Hh0072 gene, a H. pylori 11639 FucTa gene, or a H. pyloriUA948 FucTa gene.